A morphoanatomical approach to the adaptive features of the epidermis and proboscis of a marine Polychaeta: Eulalia viridis (Phyllodocida: Phyllodocidae)

Abstract

Eulalia viridis is a marine Polychaeta of the rocky intertidal that, despite its simple anatomy, is an active predator of much larger invertebrates, from which it extracts pieces of soft tissue through suction. This uncanny feeding strategy triggered the pursuit for the morphological mechanisms that enable adaptation to its environment. The evaluation of the worm anatomy and microanatomy, combining electron and optical microscopy, revealed a series of particular adaptations in the epidermis and in the proboscis (the heavily muscled eversible pharynx). Besides its function in feeding, the proboscis is the main sensory organ, being equipped with numerous sensorial papillae holding chemoreceptors. Additionally, the proboscis possesses tentacles that become exposed when the organ is everted. These provide fast release of mucus and toxins, from mucocytes and special serous cells, respectively (the latter involving both merocrine and apocrine processes), whenever contact with a prey occurs. In its turn, the epidermis provides protection by cuticle and mucus secretion and has a sensorial function that may be associated to the worm's uncommon green pigment cells. Eulalia viridis presents a series of elegant adaptive tools to cope with its environment that are evolutionarily designed to counterbalance its relatively simple body plan.

Introduction

The Polychaeta are one of the most diverse and widespread groups of aquatic animals, occupying ecological niches as distinct as deep‐sea vents to rivers and lakes. They play a very important role in the ecosystem, virtually at all levels of the food web, from symbiosis with primary producing bacteria, to nutrient recycling in sediments and predation (e.g. Hutchings, 1998; Glasby & Timm, 2008). Despite sharing the same relatively simple body plan, the extraordinary diversity of polychaetes implies specific adaptations to habitat and ecological niches at both macro‐ and microscopical levels. The functional perspective on adaptive traits reveals several important and distinctive characteristics of these organisms. Dales (1962), for instance, even differentiated several Polychaeta families according to the proboscis (also called ‘trunk’ or ‘eversible pharynx’), which shows radiated adaptation to multiple feeding and sensing strategies. Likewise, the epidermis of these animals requires special adaptations to provide protection and sensing abilities that must match the species’ ecology (for a review see Hausen, 2005). In fact, the epidermis of these animals has been revealing a complex arrangement of secretory cells whose function can vary from the release of inorganic compounds to mucopolysaccharides (Dorsett & Hyde, 1970a,b). Other types of cells, such as specialised pigment cells, are also common (see Bandaranayake, 2006).

Eulalia viridis (Linnaeus 1767) is a phyllodocid equipped with an eversible proboscis devoid of jaws or similar structures. Yet, the species is mostly carnivorous and is able to predate live and larger prey, including mussels, barnacles, gastropods and even other annelids, seemingly by extraction (via suction) of pieces of flesh (Emson, 1977; Morton, 2011; Cuevas et al. 2018). These odd features, together with its uncanny green coloration (provided by unknown green pigments), has led to some important work in the past that provided the first description of its anatomy (Michel, 1964, 1968, 1969, 1970). An effort to conjugate a relation between phenotype (behaviour included) and ecology has recently been attempted by the authors (Cuevas et al. 2018). The existence of specialised serous‐like cells in the proboscis was first highlighted by Michel (1964, 1968), who hypothesised their role in digestion. These cells were thought to secrete digestive enzymes such as trypsin, similarly to other Polychaeta like the better‐known predator Nereis virens (Michel, 1970; Michel & DeVillez, 1980). However, some of our recent findings suggest the secretion of a proteinaceous toxin, ‘phyllotoxin’, which helps to explain the predatorial behaviour of this species, as it seems to exercise an immobilising effect on soft‐bodied animals, including bivalves and other Polychaeta (Rodrigo et al. 2014; Cuevas et al. 2018). Similarly, despite the work that has been done to describe the epidermis of Polychaeta (Bubel, 1983; Tzetlin et al. 2002), little focus has been given to Eulalia. Pigment cells in E. viridis were noticed in sensorial papillae and the pharynx (Michel, 1964), and the presence of pigments and their transformation processes in the intestine have been investigated (Rodrigo et al. 2015). However, the function of pigments cells and sensory organs in the epidermis of this worm remains, to date, conjectural.

The secretion of toxins by marine invertebrates is a matter of great interest, owing to the growing potential of these compounds for biotechnological purposes. The most prominent examples are conotoxins, produced by Conus snails (for a review see Kumar et al. 2015). In annelids, the presence of peptidic toxins was first described in specimens equipped with dedicated glands, such as Glycera (Bon et al. 1985). However, detailed data from Eulalia is essentially lacking. In general, more focus has been directed on mucous secretions as a defensive mechanism than on toxin delivery in the Polychaeta (e.g. Prezant, 1980). Altogether, the current state‐of‐the‐art in Polychaeta morphology and microanatomy is mostly based on past literature that still presents many gaps regarding function. The aim of the present work is to explored the anatomical and microanatomical features of the proboscis and epidermis of E. viridis in the face of their function and adaptive value, which have enabled it to be such a successful predator of the rocky intertidal along European shorelines.

Materials and methods

Adult worms were collected from the Parede beach, Western Portugal (38°41′42″ N, 09°21′36″W), a rocky intertidal beach with large patches of barnacles and mussels upon which the worm is known to feed. The animals ranged between 50 and 120 mm in length and weighed about 250 mg.

The animals were fixed in 2% v/v glutaraldehyde (in 0.2 m sodium cacodylate buffer, pH 7.4) and Zenker's solution [2.5% mass/vol (m/v) potassium dichromate, 3% m/v mercury chloride, 1% m/v sodium sulphate and 5% v/v glacial acetic acid]. Fixation was done at room temperature for 2 h in both cases, after which worms were divided in serial sections. Fixation in glutaraldehyde was followed by washing in cacodylate buffer (3 × 15 min), overnight post‐fixation in 1% m/v osmium tetroxide (OsO₄) in 0.2 m cacodylate buffer (pH 7.4) in the dark, and washing in Milli‐Q‐grade ultrapure water (3 × 10 min). A first set of these samples was dehydrated in a progressive series of ethanol (30–100%), intermediately infiltrated with xylene and embedded in paraffin (Paraplast) wax. Paraffin‐embedded samples were sectioned (5 μm thickness) with a Jung RM2035 model rotary microtome (Leica Microsystems). A second set was embedded in Epon resin, following Luft's mixture (Sigma‐Aldrich, St. Louis, MO, USA), after being dehydrated in acetone. Intermediate infiltration was done with Epon : polypropylene oxide 1 : 2, 1 : 1 and 2 : 1 (30 min each). Semi‐thin (200–500 nm) and thin (≈ 100 nm) sections were obtained using an 8800 Ultrotome ultramicrotome (LKB Bromma).

Histological and histochemical analyses, both in paraffin and in resin sections, were performed using a tetrachrome (TC) technique based on Alcian Blue (AB) for acidic sugars (pH 0.5–2.5 to indicate sulphated and carboxylated mucins, respectively), Periodic Acid/Schiff's (PAS) for neutral polysaccharides, Weigert's iron Haematoxylin (WH) for chromatin, and Picric Acid (PA) for muscle and cytoplasm, following Costa & Costa (2012), with modifications (Costa et al. 2014; Rodrigo et al. 2015). Resin sections required permeabilisation using a saturated solution of potassium hydroxide in ethanol 100% for 5 min followed by 2 min of 2% m/v borax (sodium borate) just before staining. Other general staining procedures were applied to enhance specific details, such as Toluidine Blue (TB), Paragon, van Gieson's trichrome (VG), Coomassie Blue, Acridine Orange (AO) and special combinations such as PAS‐TB. Details of the procedures can be found in Costa (2018). Histological analyses, focused on epidermis and proboscis, in both paraffin and resin sections, were done with a DMLB model microscope adapted for epifluorescence with an EL6000 light source for mercury short‐arc reflector lamps. The microscope was equipped with A, N2.1 and I3 filters (corresponding to blue, red and green channels, respectively). All equipment was supplied by Leica Microsystems.

Thin resin sections were analysed by transmission electron microscopy (TEM). For this purpose, sections were collected onto copper or nickel mesh grids and stained with 2% m/v aqueous Uranyl Acetate and Reynold's Lead Citrate (Venable & Coggeshall, 1965). Analyses were done using a JEOL 100‐SX model TEM operated at 80 keV.

External structure was analysed by scanning electron microscopy (SEM), following the protocol described by Inoué & Osatake (1988), with modifications. In a first approach, samples were fixed in 2% v/v glutaraldehyde (in 0.2 m sodium cacodylate buffer, pH 7.4), followed by post‐fixation in 1% m/v osmium tetroxide (OsO₄) in 0.2 m cacodylate buffer (pH 7.4). The strategy yielded good structural detail, but the epidermis and overlying cuticle became wrinkled. This issue was solved using Zenker's solution as fixative, albeit with some loss of detail. After fixation, all samples intended for SEM were dehydrated in a progressive series of ethanol (30–100%), infiltrated with tert‐butanol (3 × 15 min at 40 °C) and allowed to freeze overnight at 4 °C. The tert‐butanol was then sublimated under vacuum till complete dryness. Finally, the specimens were mounted on an aluminium disc for gold coating using a JEOL JEE‐400 vacuum evaporator. Analyses were performed in a JEOL JSM‐5400 Scanning Microscope operated at 15 and 35 keV.

Results

General anatomy

In the head, or prostomium, SEM revealed a single antenna, one pair of simple eyes, one pair of ciliated nuchal organs positioned dorsolaterally, and two pairs of palps, but no jaws or similar structures (Fig. 1A). Four pairs of long tentacular cirri were found in the subsequent segments: one pair in the first segment, two pairs in the second and one pair in the third (Fig. 1A). The worm's retractile pharynx, the proboscis (‘trunk’), was externally covered with sensorial papillae (Fig. 1B). The eyes were found to possess lenses and to be deeply embedded within the head, being covered with skin cuticle. The eyes are internally lined with a layer of melanocyte‐like cells holding brown‐blackish pigments (Fig. 1C), which is in accordance with the observations by Whittle & Golding (1974). Eulalia viridis, like most Errantia, has one pair of locomotor parapodia per segment, composed of a single lobe, the neuropodium, with a central cylindrical structure supported by a central chaeta, named aciculum, surrounded by numerous small chaetae that end in a hook (Fig. 1D). From the centrum of the neuropodium, two cirri emerge, a small ventral cirrus and a larger elongated and flattened dorsal cirrus that displays numerous mucocyte‐like cells arranged transversally (Fig. 1E).

Figure 1.

General anatomy of the Polychaeta Eulalia viridis. (A) First segments of the worm imaged with SEM, showing four pairs of long tentacular cirri (tc) and the parapodia (pd) in the remaining segments (seg). The worm is fitted with a single antenna (an), one pair of eyes (ey) and one pair of ciliated nuchal organs (no). Two pairs of palps (pal) are located near the mouth, through which the proboscis is extended (pr). Papillae are absent from this image due to incidental loss of proboscidial epidermis. (B) Tip of the semi‐extended proboscis (pr), highlighting its numerous papillae (pap) covering its fibrous epidermis (SEM). (C) Resin section (Toluidine Blue stain) across one of the eyes (ey) of the worm, showing the densely packed layer of melanocyte‐like pigment cells (pc) underneath the photoreceptor layer. (D) SEM image of parapodia, showing a pair of parapodia (pd) per segment (seg). Note chaetae (ch) emerging from neuropodia, the small ventral cirri (vc), and the larger, elongated and flattened dorsal cirri (dc). (E) Transversal section of the dorsal cirrus of a parapodium, highlighted with a circle in (D) (paraffin section stained with van Gieson's trichrome), exhibiting the particular distribution of mucus cells (mc).

Epidermis

The epidermis (Fig. 2) covers the entire body, with the exception of the proboscis. It is made up of a pseudostratified epithelium formed by clearly distinct cell types. This epithelium is protected by a collagenous cuticle and an external epicuticle. Cuticle and epicuticle were found to be reactive to Periodic Acid/Schiff's (PAS‐positive) and to Alcian Blue (AB‐positive), respectively. Histology showed that the thickness of the epidermis varies between body areas, being thicker in the ventral area and around parapodia. From the histochemical appraisal, three main types of cells were identified: supportive cells (responsible for the secretion of cuticle and epicuticle), mucocytes (mucous‐secreting) and green pigment cells. Basal (replacement) cells could be distinguished attached to the basal lamina, easily spotted due to their pyramidal shape (Fig. 2A, inset).

Figure 2.

Cuticle and epicuticle secretory cells in epidermis. (A) Resin section (tetrachrome stain) across the ventral epidermis of Eulalia viridis, showing the granules in supportive cells responsible for cuticle production (ctc), which presented the same PAS‐positive (pink) reaction of the cuticle (ct). Similarly, granules of supportive cells responsible for epicuticle production (ectc) and epicuticle (ect) shared the same AB‐positive (blue) reaction. Inset: TEM image (Toluidine Blue stain) of the epidermis showing three basal cells (bc); the third has already started to differentiate into a pigment cell (pc). (B) Cuticle‐secreting cells presented Acridine Orange fluorochrome (AO) metachromasia (yellowish‐green), whereas epicuticle secretory cells were red‐fluorescent (AO orthochromasia), seen in this image from a paraffin section. Inset: Detail of the metachromatic cuticle and orthochromatic epicuticle.

The supportive cells were thin and elongated, had a distinctive crypt opening to the outside through a thin channel, and numerous granular inclusions that shared the histochemical signature of cuticle and epicuticle, i.e. PAS‐positive (strong pink) and AB‐positive (blue), respectively (Fig. 2A). The latter case is indicative of acid sugars of mucins or the glycocalyx bound to the microvilli of epithelial cells. The fluorescence signature of supportive cells under ultraviolet (UV) light, after AO staining, also confirmed their association to the cuticle and epicuticle (Fig. 2B). Consequently, cuticle and inclusions associated with supportive cells were AO metachromatic (yellowish‐green), whereas the epicuticle was AO orthochromatic (reddish), as shown in Fig. 2B (inset). The supportive cells were disseminated throughout the epidermis, albeit in larger numbers in the ventral area.

Mucocytes were present in large numbers and were evenly distributed along the epidermis. The cytoplasm was mostly occupied by saculi whose size and histochemical signature was used to distinguish between the three main maturation stages of the cells (Fig. 3A). At stage 1, mucopolysaccharides in saculi were TB metachromatic (reddish), electron‐dense (Fig. 3B) and reactive to PAS as well. Cells at this stage were consistently closer to the basal lamina of the epithelium. At stage 2, mucus cells had more TB‐orthochromatic saculi (blue‐purple), albeit with varying colour intensity and size. The saculi became then mostly electron‐transparent (Fig. 3C) and more reactive to AB, regardless of pH, indicating a likely blend of carboxylated and sulphated mucosubstances. Finally, stage 3 mucus cells were only mildly AB reactive and contained large and electron‐transparent saculi (Fig. 3D). Mucus cells in stages 2 and 3 were seen to possess crypts that formed narrow channels toward the surface, just like cuticle‐ and epicuticle‐secreting cells (Fig. 3E). Mucin‐producing cells were noted to have an important structural function in parapodia as well. At the base of each parapodium there was a structure containing numerous mucus cells in the three stages. Additionally, a two‐layer supportive structure was observed in the dorsal cirrus of the parapodia, each layer being formed by densely packed mucus cells at stage 2 (cf. Fig. 1E). In both cases, these mucocytes were devoid of conspicuous crypts.

Figure 3.

Mucus cells in the epidermis of Eulalia viridis. (A) Resin section (Toluidine Blue) showing the three stages of mucus cell maturation: stage 1 (mc1), stage 2 (mc2) and stage 3 (mc3). (B) Stage 1 revealed electro‐dense saculi (sa) in TEM. (C) Saculi in stage 2 were mostly electron‐transparent (TEM). (D) In stage 3, the cells possessed larger and electron‐transparent saculi (TEM). (E) SEM image of the epidermis, fixated with glutaraldehyde, showing openings (op), or crypts, of mucus and cuticle‐ or epicuticle‐producing supportive cells.

Pigment cells did not appear to have a secretory function but were seemingly responsible for the production and/or transformation and storage of pigments in granule‐like structures. Pigment cells appeared in large numbers throughout the epidermis and were easily identified without the use of dyes (Fig. 4A). They possessed heavily electron‐dense granules that remained green regardless of histological dye, in both paraffin and resin sections, as previously described by Rodrigo et al. (2015). The pigment cells were compressed between the remaining epidermis cells, to which they were bound with ‘cuff’ links. Nuclei were positioned basally. The cells were attached to the cuticle by microvilli, forming an apical branch‐like structure (Fig. 4A, inset). They were also found to bear several cilia that protruded through the cuticle (Fig. 4B).

Figure 4.

Typical green pigment cells (pc) that give the worm its distinctive coloration. (A) Green pigment granules (gp) inside pigment cells (pc) visible in an unstained paraffin section. Inset: TEM image of the apical section of a pigment cell bearing electron‐dense intercellular ‘cuffs’ (c) of adherens junctions. The cell is anchored in the cuticle with numerous microvilli (mv). ct, cuticle; ect, epicuticle. (B) TEM image showing the sensorial cilia (sci) of pigment cells protruding through the cuticle (ct) and epicuticle (ect). Evidence for a cuff (arrow) connecting a pigment cell (pc) with a mucocyte at the left. mv, microvilli.

Proboscis

The proboscis is a heavily muscled eversible pharynx that was found to be lined with an integument different from the epidermis, as it was essentially fibrous and elastic, and bore numerous sensorial papillae (Fig. 5). When the proboscis was extended, the papillae were exposed to the environment, as were a series of tentacles arranged in a crown around the gut opening (Fig. 5A). The proboscis was composed of two main layers, inner (mostly muscular) and outer (epidermis) (Fig. 5B). These layers were not bound by connective tissue. Instead, the space between them allowed retention of coelomic fluid during the extension process, generating hydrostatic pressure.

Figure 5.

Microanatomy of the proboscis in Eulalia viridis. (A) Longitudinal section across the retracted organ. The outer layer is formed by a fibrous epidermis (ep), clearly distinct from the epithelium of the main body skin. The integument presented numerous sensorial papillae (pap) and a thin muscle layer underneath. The inner layer, inside which the specialised tentacles for toxin delivery are shown (arrow), forms a rosette attached to the strong pharynx musculature (phm). Paraffin sample, tetrachrome stain. (B) Transversal section of the proboscis. inl, inner layer where the pharynx cavity (ph) is highlighted; outl, outer layer; pap, papillae; phm, pharyngeal musculature; tm, transversal muscle bundles. Paraffin section, tetrachrome stain. Inset: Magnification of nervous fibres (nf) and nervous cord (nc) connected with all the papilla. Paraffin sample, tetrachrome stain. (C) SEM image (sample treated with Zenker's fixative) of papillae on the surface of the proboscis. Cilia and other details are not visible due to their reduced size and sample treatment with mercury chloride, which enhances structure integrity (note absence of wrinkling), albeit at the cost of some detail. (D) Transversal resin section of papillae highlighting mucus cells (mc), cuticle‐producing supportive cells (ctc) and pigment cells (pc). Toluidine Blue. Upper inset: Chemoreceptor on a papilla. Note high concentration of pigment cells (pc) and the crown of cilia (cl). Resin section, tetrachrome stain. Lower inset: Large openings (arrow) in papillary chemoreceptors (SEM). ct, cuticle; ect, epicuticle; op, secretory cell openings/crypts.

The outer layer is essentially comprised of cuticle, epicuticle, fibrocytes and fibres. The papillae, which displayed a complex arrangement of different cells plus connective and nervous fibres (Fig. 5B, inset), formed conspicuous rounded domes on the epidermis (Fig. 5C). They were also lined by epicuticle and cuticle. The major cell types in papillae could be identified as mucocytes, pigment cells and the supportive cells responsible for the production of cuticle and epicuticle (Fig. 5D). Each sensorial papilla had chemoreceptors that bore pigment cells and cilia (Fig. 5D, upper inset) around openings (Fig. 5D, lower inset). The inner layer of the proboscis was lined internally by the pharynx epithelium, which consisted of a pseudostratified layer of pigment and serous cells, covered with cuticle and epicuticle as well (Fig. 6A,B). Special serous cells (toxin‐secreting) were densely packed at the base of the tentacles mentioned above. The tips of the tentacles were chiefly composed of mucus cells, mostly in stages 2 and 3, with visible crypts (Fig. 6C). Secretion of mucus formed domes that were easily detected by SEM (Fig. 6D).

Figure 6.

Description of the proboscidial inner layer and tentacles at the edge of the pharynx. (A) Longitudinal section (paraffin) of the pharynx (internal) epithelium with serous cells (sc), which are mildly PAS‐positive and stained by Picric Acid as well. pc, pigment cells; phm, pharynx musculature. Resin section, tetrachrome stain. Inset: The vesicles of serous cells (sc) were densely stained by Coomassie Blue, indicating proteinaceous materials. (B) The base of a tentacle (tb) is characterised by densely packed serous cells (sc) intercalated with a few pigment cells (pc). Resin section, Toluidine Blue. (C) Longitudinal section across the tentacles, which are divided in a basal area (tb) with numerous serous cells (sc) and neuronal fibres (nf), and the tentacle tip (tt), with few serous cells and numerous mucocytes (mc). Paraffin sections, tetrachrome stain. (D) SEM tentacle surface with visible domes extruding from secretory cells (arrows).

Toxin‐secreting cells were easily identifiable by their idiosyncratic chalice‐like shape (Fig. 7A). The apical part of the cell, closer to the cuticle, presented numerous electron‐dense vesicles lined by a double membrane (Fig. 7A, inset). The nucleus was found closer to the basal lamina, as well as a well‐developed rough endoplasmic reticulum (Fig. 7A) and numerous mitochondria. Vesicles were densely stained by Coomassie Blue, indicating proteinaceous material (Fig. 6A, inset). The protein vesicles were also PAS‐positive (Fig. 6A) and TB orthochromatic (Fig. 6B). Different staining intensities revealed cell maturation stages and the loss of cellular material after toxin secretion (Fig. 7B,C). Whereas multivesicular bodies (MVBs) were a common feature in maturing toxin cells (Fig. 7C), mature cells held mostly electro‐dense and Coomassie Blue‐positive vesicles, whose size prevented them from crossing the cuticle (Fig. 7D,E).

Figure 7.

Toxin cells and toxin secretion processes. (A) Toxin cells (TEM micrograph) showing their typical chalice shape, electron‐dense (mature) toxin vesicles (tv) and a well‐developed rough endoplasmatic reticulum (rer). Inset: Higher power TEM magnification of the mature toxin vesicles (tv) with double membrane. ct, cuticle; ect, epicuticle. (B) Semi‐thin section (resin) of the pharynx epithelium in the proximity of the tentacles shown in Fig. 5A, presenting mature (mtc) and immature (itc) toxin cells (Toluidine Blue stain). ct, cuticle; ect, epicuticle; phm, pharyngeal musculature. (C) Detail of a maturing toxin cell (mtc) with multivesicular bodies (mvb) and fully mature toxin vesicles (tv), the latter holding electro‐dense, highly concentrated, proteinaceous materials (TEM). Maturing vesicles are essentially multivesicular bodies that may form exosomes or progress into mature toxin vesicles. pc, pigment cell. (D) Merocrine secretion (via exocytosis) of exosomes (ex) (TEM). ct, cuticle; tv, mature toxin vesicles. (E) Merocrine secretion in mature toxin cells (TEM). ct, cuticle; ect, epicuticle; ex, exosomes; tv, toxin vesicles. (F) Microvilli‐like expansions (arrows) in the apical membrane of toxin cells formed during apocrine secretion (TEM). Inset: Detail of the apocrine and merocrine secretion processes occurring simultaneously at the surface of the epicuticle. Note profusion of exosomes and more electro‐dense apocrine blebs (arrow). ct, cuticle; ect, epicuticle; tv, toxin vesicles.

Secretion from toxin cells was found to occur by two processes. Under steady‐state conditions, exocytosis was a continuous process from maturing cells (merocrine secretion), with exosomes being released mostly from MVBs (Fig. 7D,E). However, sections from the everted proboscis revealed an apocrine‐like process in mature cells, during which the apical portion of the cytoplasm and mature toxin vesicles were subjected to internal and external pressure, generating evaginations that cross the cuticle (Fig. 7F). These microvilli‐like expansions contain the same electro‐dense materials found in mature toxin vesicles (Fig. 7F, inset). The two processes are summarised in Fig. 8, highlighting that the apocrine process is seemingly responsible for rapid secretion of toxins and associated molecules when the tip of the everted trunk contacts a target.

Figure 8.

Simplified diagram illustrating the two main toxin secretion pathways. Toxins and accompanying proteinaceous materials are initially produced in the rough endoplasmic reticulum (RER), concentrated in the Golgi apparatus (GA) and allocated in multivesicular bodies (MVBs). These will either participate in the formation of exosomes or further develop into vesicles with high toxin concentration. In the first case, secretion occurs via simple exocytosis (merocrine process), when MVBs merge with the apical membrane of the cells. This process is continuous, occurring in steady‐state maturing and mature toxin cells. When fully mature toxin cells are subjected to pressure, they will rapidly release the stored, highly concentrated materials via an apocrine process. The internal pressure derives from increasing size and number of toxin vesicles. Extracellular pressure is caused by a combination of trunk muscle contraction, increased hydrostatic pressure when the proboscis is everted, and direct contact between the tip of the proboscis (where toxin‐delivery tentacles are located) and prey. The compression leads to the formation of microvilli‐like expansions that rapidly protrude through cuticle and epicuticle.

Discussion

Eulalia viridis is an active predator that, like the remaining members of the family Phyllodocidae, is characterised by its strong muscular proboscis, although it is devoid of jaws or similar structures. This latter aspect is compensated by the ability to immobilise prey (such as mussels and other polychaetes) and extract a portion of soft tissue. Immobilisation requires only the direct contact between the specialised tentacles and prey as they are projected outward when the proboscis is fully everted (for a description of the worm's predatory behaviour see Cuevas et al. 2018). The process is assisted by the particular features of the proboscis that make it the species’ main sensory organ. Altogether, the animal's adaptations to its feeding strategy are well reflected in its anatomy and microanatomy.

The proboscis alone is a major adaptive trait: (i) it is strongly muscular, allowing action as a suction pump; (ii) it has a dense battery of sensory organs, specifically chemoreceptors, and (iii) the copious production of mucins is linked to the secretion of toxins and proteolytic enzymes. The suction action of the proboscis and the secretion of mucins and enzymes that were only briefly addressed by Michel (1970), have been recently detailed by Cuevas et al. (2018). Despite these previous works, there is still little information on Polychaeta concerning their feeding strategies. Nonetheless, the basic morphoanatomy and function of the proboscis appear to be well conserved among the Polychaeta (Dales, 1962). On the other hand, Eulalia presents the overall typical epidermis of an errant polychaete, i.e. thicker in the ventral side, with a sturdy cuticle. This arrangement is ideal for scavenging through the rocky intertidal (see the review by Hausen, 2005). The epidermis that lines the proboscis is, nonetheless, morphologically very distinct from the epidermis that covers the rest of the animal's body, and will be dealt with separately.

Two main functions are attributed to the skin epidermis. The first is protection and is adjuvated by the secretion of cuticle and mucus. The second is sensorial, albeit more elusive in this species. Sensing is probably associated to the same pigment cells that give the worm its characteristic green colour. Due to the absence of blood vessel networks (in parapodia, inclusively), the role of the epidermis as ‘gills’ (Tzetlin et al. 2002) is excluded in this species. The secretion of simple substances via epidermis (e.g. acting as pheromones) cannot be entirely dismissed. Nonetheless, the existence of multicellular glands, such as the spiral glands of Nereis (Dorsett & Hyde, 1970b), is overruled.

Besides the usual sensory organs in the prostomium (antennae, nuchal organs, cirri and eyes) used by polychaetes to sense the environment (for a review see Purschke, 2005), Eulalia uses its extended proboscis as the main sensory organ to scan the environment as the worm scouts through rocks, mussels and barnacles (Cuevas et al. 2018). When the worm encounters a prey, the proboscis is everted via muscular action and increased hydrostatic pressure of the coelomic fluid, then externalising the specialised tentacles (Fig. 5. As these structures touch the prey, they administer a proteinaceous toxin (or toxins) accompanied by digestive enzymes, referred by us as ‘phyllotoxin’ (Rodrigo et al. 2014; Cuevas et al. 2018). The exact nature of these substances is still being investigated. However, they seem to immobilise prey while partially digesting the tissue at the contact area, thus enabling extraction of tissue via suction in absence of jaws or analogous structures. The serous cells in the pharynx, particularly those densely packed near the base of the tentacles, are seemingly involved in the production of the proteinaceous components of the toxin. The mucus, secreted by the mucocytes that are present throughout the animal and present in large numbers in the tips of tentacles, acts like the conveying vehicle (i.e. the carrier fluid) of toxins (cf. Fig. 6). Michel (1968) hypothesised an ‘adhesive’ function for the tentacles (hitherto referred to as ‘grosse papille’) because of the mucus secretions. However, in the aforementioned work, neither the adhesive properties of mucus or the existence of toxin secretions was then suspected. The function of these special tentacles is assisted by a dense complex of neuronal fibres (Fig. 5B, inset), which could indicate mechanoreception. It must be noted, though, that this sensorial function is not well understood in marine invertebrates, particularly in the Annelida. Altogether, the role of the proboscis in sensing the environment makes it a crucial feature for an active forager. The sensorial papillae (the ‘petite papille’ referred by Michel, 1964) are attached to the fibrous outer epidermis of the proboscis. The associated chemoreceptors (taste buds) contain many of the species’ uncanny pigment‐bearing cells, which adds to the hypothesis that the green pigments may be associated to sensory functions, as discussed further below.

Toxin secretion has been hypothesised for other phyllodocids, particularly in species with specialised glands, such as in some Glycera (Michel, 1970; Bon et al. 1985; Böggemann et al. 2000). In E. viridis, however, there are no complex multicellular glands in the proboscis or epidermis. Even though serous cells lining the epithelium of the pharynx have been associated to the production of digestive enzymes (Rodrigo et al. 2015), these cells, especially those located in the vicinity of tentacles, are thus likely associated to toxin secretion (see Cuevas et al. 2018).

Michel (1969, 1970) hypothesised that the secretion of proteolytic enzymes in Polychaeta is essentially merocrine, without, however, clearly identifying how the process occurs. The present findings suggest that secretion in serous toxin cells is merocrine during resting, as seen in similar vertebrate cells (Hussein et al. 2015). Nonetheless, when the worm makes contact with prey, apocrine secretion is set in motion, caused by changes in internal cell pressure, which enables faster release of highly concentrated products (cf. Fig. 8). For a review of the secretion of exosomes and related processes see van Niel et al. (2018). The ability to rapidly deliver the complex cocktail of toxins, permeabilising enzymes and other substances that make up animal poisons is an advantage for a small predator that reaches for its targets using the proboscis as it is moved to sense the environment. In fact, our previous work recorded Eulalia immobilising another Polychaeta simply by repeated contact with the tip of the fully everted proboscis, at precisely the site where the toxin‐delivery tentacles are present (Cuevas et al. 2018).

The coexistence of both apocrine and merocrine secretion has already been reported to occur in gland cells of higher order vertebrates, from eyes to reproductive organs (Groos et al. 1999; Hussein et al. 2015). Rather than shifts in osmotic pressure, as proposed by Michel (1969), apocrine secretion in serous cells of the proboscis is triggered by a combination of: (i) mechanical pressure exerted by contact with prey; (ii) increased hydrostatic pressure within the proboscis; and (iii) contraction of adjacent musculature (e.g. during suction). Serous cell turgidity caused by water intake was not observed. Additionally, increased cell‐cell and cell‐cuticle spacing, which could indicate fluid retention, was noticed only when serous cells were empty. However, these features may result from fixation artefacts.

The epidermis of E. viridis is a well‐developed organ that suits the habits of an Errantia, which implies not only defence against macro‐ and microorganisms but also sensing of the environment and protection during locomotion. In addition, it is possible that the epidermis is involved in chemical signalling, similarly to the alarm cells of fish (Chivers et al. 2007), as animals in captivity have been observed to react negatively to the proximity of an injured conspecific (own observation). The main cells in the epidermis of E. viridis are involved in the secretion of cuticle, epicuticle and mucus. The distribution and number of these cells varies between body regions. In the ventral area, more subject to abrasion from the rocky substrate, the epidermis is thicker, which is accompanied by increased numbers of supportive cells responsible for the production of cuticle and epicuticle, consistent with reports from other Phyllodocida (Tzetlin et al. 2002). In parapodia, mucus cells are abundant and have important roles in structure and locomotion (lubrication).

The co‐existence of supportive cells responsible for the production of cuticle (PAS‐positive) and epicuticle (AB‐positive) has been described for several polychaetes (e.g. Dorsett & Hyde, 1970b; Richards, 1974; Bubel, 1983). Their ubiquity led to them being simply named ‘epidermal cells’ in the past (Krall, 1968; Burke & Ross, 1975). Our results are in accordance with the findings of Bubel (1983) on the polychaete Pomatoceros zamarckii; they suggested that cuticle and epicuticle originate from cytoplasmic granules arising from the Golgi apparatus. The collagenous nature of the cuticle of the Phyllodocida (Michel, 1969; Kimura, 1971; Hausen, 2005) is in accordance with the fibre arrangement typical of the Errantia (Westheide & Rieger, 1978; Storch, 1988). This pattern, which in the past was associated to body size, is now thought to be linked to mobility, as it safeguards both resistance and elasticity (see Goodman & Parrish, 1971; Westheide & Rieger, 1978). In addition, Bubel (1983) maintained that cuticle and epicuticle are supported by the microvilli of epidermal cells, a role that, in Eulalia, is seemingly secured by pigment cells. Due to the potential role of these cells in sensing, it is possible that the function of microvilli extends to sensory roles as well, as discussed below. The junction between epidermis and cuticle in annelids remains unknown, as pointed out since the work by Krall (1968), and it is possible that it displays significant variability between taxa.

Mucus cells, in their turn, have been extensively studied in invertebrates (Storch & Welsch, 1972a), including both errant and sedentary polychaetes (Mastrodonato et al. 2005, 2006), as well as phyllodocids (Ushakov, 1972). The function and processes underlying mucus secretion in Polychaeta are, however, highly variable according to species’ ecology. Specifically, mucocyte function varies greatly from coastal sedentary (see Storch, 1988; Hausen, 2005 and Mastrodonato et al. 2005 for more details) to errant Polychaeta, especially those inhabiting intertidal areas. In this latter case, mucosubstances are involved in thermal regulation (Ushakov, 1972), prevention from desiccation, lubrication for locomotion, egg protection, and as part of defence and predation mechanisms (Storch & Welsch, 1972b; Prezant, 1980; Storch, 1988; Mastrodonato et al. 2006). All these functions can thus be justifiably applied to E. viridis. To these are added the structural function of mucosubstances in parapodia (recall the stage 2 mucus cells in dorsal cirri) and as carriers of toxins, which is secured by stage 3 mucus cells in the tentacles.

The ultrastructure of mucocytes in E. viridis agrees with previous research on aquatic invertebrates (McKenzie & Hughes, 1999; Mastrodonato et al. 2005). Moreover, the present findings agree with earlier postulates of the existence of two major type of mature mucocytes in Polychaeta (Storch & Welsch, 1972a; Storch, 1988). Besides differences in mucus chemistry, in Eulalia, stage 2 cells extrude the contents of saculi, whereas in stage 3, mucins remain internalised. Our findings suggest that both stage 2 and stage 3 mucus cells arise from cells identified as stage 1 (see Fig. 3). This demonstrates the link and ontogeny of mucus cells proposed by some authors (Storch & Welsch, 1972a; Bubel, 1983; Mastrodonato et al. 2006), in contradiction to the hypothesis that these constitute, in fact, different cells (Ushakov, 1972).

It has long been suspected that certain types of microvilli‐bearing cells in the epidermis of polychaetes and other annelids have a sensorial function, being able, inclusively, to trigger the secretion of substances (such as mucins) from adjacent cells (Lent, 1973; Schlawny et al. 1991). The microvilli appear to be the key ultrastructural feature, as they transverse the cuticle and come in direct contact with the environment (revised by Hausen, 2005 and Purschke, 2005). It may thus be inferred that the special microvilli‐bearing pigment cells in the epidermis and proboscis of Eulalia possess a sensorial function. In the papilla, in particular, pigment cells form part of chemoreceptors (which was first reported by Michel, 1964, albeit without a functional explanation), reinforcing the notion that they play a role in sensing the environment. Also, besides the presence of pigment vesicles, pigment cells resemble the mechanoreceptors described for the epidermis of other species of Polychaeta, in particular with respect to shape, positioning, distribution, microvilli surrounding special cilia, and the electro‐dense ‘cuffs’ of adherens‐like junctions (Jouin et al. 1985; Schlawny et al. 1991). It should be noted that mechanoreceptor cells in the Annelida can be extremely diversified (see Purschke, 2005). Altogether, it is most likely that the sensorial function of the epidermis is triggered by a combination of mechanical and biochemical stimulation in microvilli and cilia. However, other functions for pigment cells should not be discarded. Even though the worm's bright green colour is unlikely to act as camouflage, these uncanny green pigments may offer protection against UV light, similarly to melanins (see Riaux‐Gobin et al. 2000). Even though the nature of the green substances has not yet been disclosed, they most likely result from haem breakdown (Lederer, 1939; Kennedy, 1975; Rodrigo et al. 2015). Another possible function of these pigments can be aposematism, i.e. the association between the animal's bright green coloration and its toxicity, therefore warning off attackers (for a review see Bandaranayake, 2006).

Conclusions

Eulalia viridis compensates for its simple morphoanatomy with a series of subtle microanatomical features that serve as an adaptation to its environment and suit its predatorial behaviour. These include the sensorial papillae of the proboscis, which, being fitted with taste buds, are the main sensorial organs of the worm, without prejudice for eyes and cirri in the Prostomium. Special pigment cells in papillae and epidermis likely act as mechanoreceptors. This latter feature may represent a major adaptive trait, as it allows a fast, involuntary response to external stimuli. These adaptations, combined with the differentiated secretion of mucus, cuticle and toxins (the latter of which able to be discharged rapidly), render the species a very effective predator of the rocky intertidal, where it forages for invertebrate prey, including larger molluscs and even other Polychaeta, including its conspecifics. It has thus been demonstrated that a large part of the evolutionary success of protostomes, in spite of their simple body plan, is due to subtle anatomical features that establish an inconspicuous but very efficient link between the behaviour of the animals and their habitat.

Authors’ contribution

A.P.R. and C.M. performed the laboratory work and prepared the manuscript with relevant input from A.P.M., M.H.C. and P.M.C. A.P.M. supported all analyses involving electron microscopy and P.M.C. designed the experiments and supervised the work. The authors declare that there are no conflicts of interest.