© microscopy societyof america 2014 a study on the...

Microsc. Microanal. 21, 91–101, 2015doi:10.1017/S143192761401352X

© MICROSCOPY SOCIETYOF AMERICA 2014

A Study on the Digestive Physiology of a MarinePolychaete (Eulalia viridis) through MicroanatomicalChanges of Epithelia During the Digestive CycleAna P. Rodrigo, 1 Maria H. Costa, 1 António Pedro Alves de Matos,2 Francisco Carrapiço, 3 andPedro M. Costa 1,*

1MARE—Marine and Environmental Sciences Centre/IMAR—Instituto do Mar, Departamento de Ciências e Engenharia doAmbiente, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica, Portugal2Centro de Estudos do Ambiente e do Mar (CESAM)/Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Quinta daGranja, Monte de Caparica, 2829-511 Caparica, Portugal3Centro de Biologia Ambiental (CBA), Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa,1749-016 Lisboa, Portugal

Abstract: As for many invertebrates, the gut of marine polychaete species has key physiological functions.However, studies integrating microanatomical descriptions with physiological processes are scarce. The presentinvestigates histological, histochemical and cytological changes in the alimentary canal during the digestive cycleof the marine annelid Eulalia viridis, a species that combines opportunist scavenging, predation and cannibalisticbehavior. The gut is comprised of an eversible pharynx, esophagus, intestine and rectum. Three main phases ofdigestion were identified, namely, resting/secretory, absorptive and excretory. The intestinal epithelium iscomplex and exhibited the most significant changes regarding intracellular digestion, excretion and storage.Conversely, the pharynx and esophagus were chiefly important for enzyme secretion. The results also indicate theexistence of two distinct types of secretory cells in the intestine, with likely distinct physiological roles. Somesimilarities have been found between the intestinal epithelia and the molluscan (especially cephalopod) digestivegland, as, for instance, the shedding of apical corpuscles by digestive cells at posterior stages of digestion.The findings indicate that the digestive process in this worm is complex and related to the many physiologicalroles that cells need to play in the presence of reduced organ differentiation.

Key words: histology, histochemistry, phyllodocidae, gastrodermal cell types, digestive cycle

INTRODUCTION

Marine polychaetes constitute the most diverse group withinAnnelida, comprising about 80 known families that radiatedabout 500m.a. ago and are phylogenetically linked to stemannelids (see Bartolomaeus et al., 2005; Weigert et al., 2014).These animals play an important ecological role in marinefood chains as prey, predators, filter-feeders, or scavengersand are likewise important bioturbators of aquatic sediments asdeposit-feeders. As such, their multiple ecological niches arereflected in significant deviations from the standard polychaeteanatomy and physiology, resulting in high phenotypic variationeven within families or species (see, e.g., Tzetlin & Purschke,2005). However, in spite of their acknowledged importance,there are many gaps in the knowledge on the physiology ofthese organisms. In fact, even though the basic structure of thedigestive system structure is documented for some polychaetespecies (see Saulnier-Michel, 1992, for a review), there is stillmissing information in the knowledge of the microanatomy ofthe digestive epithelia, with particular respect to the changesalong the digestive, secretory and excretory cycles. It must be

emphasized that the alimentary canal, particularly the intestine,likely has several vital functions, a feature that, together withintracellular digestion, tends to fade in most higher-orderanimals. As a fact, in most marine invertebrates, especiallyprotostomes, the digestive epithelia exhibit complex changesduring digestion, implying structural alterations, changesin biochemical composition and enzymatic machinery, andgeneral responsiveness of the epithelium. However, suchchanges are far better described in the molluscan digestivegland, especially in cephalopods (e.g., Semmens, 2002; Costaet al., 2014), than for any other group of marine invertebrates.

Usually, the polychaete alimentary canal is divided intothree parts: foregut, midgut and hindgut (Saulnier-Michel,1992; Jenkins et al., 2002). The foregut includes the buccalorgan and the esophagus while the midgut allocates the sto-mach (when present) and the intestine. The hindgut comprisesthe posterior most regions of the gut, which may or not bedifferentiated into a rectum (Jenkins et al., 2002). However, it isthe buccal organ, located in the foregut, which reveals the mostsignificant interspecific variation, according to feeding habits,forming, for instance, an eversible pharynx (proboscis) or evenbeing absent from filter-feeding groups (Tzetlin & Purschke,2005). Most reports describe the polychaete intestine as atube-like structure, where the main cell type is the secretory cell*Corresponding author. [email protected]

Received May 31, 2014; accepted October 10, 2014

mailto:[email protected]

(Tzetlin et al., 1992; Tzetlin & Purschke, 2005). In somepolychaete species the presence of specialized absorptive andsecretory cells has been previously described and, in somecases, the latter have been divided into serous and mucous-secreting cells (Saulnier-Michel et al., 1990). No reportswere found on the relationship between cell types and thephysiology of polychaete digestion.

The marine annelid Eulalia viridis (Polychaeta: Errantia,Phyllodocidae) inhabits rocky shores, especially mussel beds, inintertidal areas, seeking protection during high tides under-neath clumps of mussels (Emson, 1977). Eulalia viridis isbelieved to be an opportunist scavenger, since scavenging(Morton, 2011) and simultaneous detrivore and predator(Emson, 1977; Fauchald & Jumars, 1979) behaviors have beenreported. It is acknowledged that E. viridis feeds mostly onbarnacles, mussels and other worms of the same species(Emson, 1977; Morton, 2011). The worm, as in other phyllo-docids, makes use of its proboscis for collecting food itemsthrough suction. Unlike many preying polychaete species theydo not have jaws and rely on strong axial musculature typical ofthe family (see Michel, 1964; Tzetlin & Purschke, 2005). Oddlyenough, herbivore feeding has never been described for thisspecies, expected for its conspicuous bright green color (Costaet al., 2013). Moreover, as for polychaetes in general, studies onthe relation between physiology and microanatomical changesalong the digestive process of this species are still lacking, withthe exception of a study on the pharynx that compared fastingand satiated animals (Michel, 1969a). Detailed microscopystudies on the species concern spawning, reproduction (Olive,1975; Rouse, 1988), microanatomy of the pharynx (Michel,1964, 1969a), intestine (Michel, 1969b) and epidermis (Costaet al., 2013) have been conducted. There are general anatomicaldescriptions of the digestive tract of phyllodocids, which followsthe basic annelid plan, although they lack a differentiated sto-mach (Saulnier-Michel, 1992; Tzetlin & Purschke, 2005).

As for marine invertebrates in general, detailed histolo-gical studies are either absent or limited to a few key, studies.Histochemical techniques hold the advantage of being able todemonstrate the presence, distribution and fate of moleculesin small tissue samples, for the dual purpose of elucidatingboth structure and physiology. As such, the present worksurveys the physiology of the digestive process in a marinepolychaete characterized for its opportunist feeding habits bycombining histology, histochemistry and electron micro-scopy. Specifically, it: (i) characterizes E. viridis intestinalepithelium regarding structure and cell types plus respectivefunctions; (ii) identifies microscopic changes in the gut epi-thelia during digestion, and (iii) identifies and localizes themain substances and processes involved in digestive andexcretory processes, from intracellular digestion to storageand elimination of physiological by-products.

MATERIALS AND METHODS

Animal collectionAdult and subadult E. viridis (50–120 mm in length,≈250 mg total weight) were collected from a rocky intertidal

shore located at the Western Algarve coast, SW of Portugal(37°22′57.81′′N, 8°49′30.71′′W). The animals were reared inthe laboratory until processed for analyses, in a mesocosmenvironment with recirculation and periodic water changes.The mesocosm included live mussels and barnacles of whichthe worms were confirmed to prey on, in accordance to thefindings of Olive (1975).

Sample preparationWhole organisms were fixed using several solutions: Bouin’s(10% v/v formalin and 7% v/v acetic acid picric acid addedto saturation); Carnoy’s (absolute ethanol, chloroform andglacial acetic acid, 6:3:1) and 2% v/v glutaraldehyde (in 0.2 Msodium cacodylate buffer, pH 7.4). Fixation in the twoformer solutions was at room temperature for 24 h, followedby washing in Milli-Q (MQ) grade (18.2 MΩ cm) ultrapurewater (3 × 1 h) for samples fixed in Bouin’s and in absoluteethanol (3 × 1 h) for samples fixed in Carnoy’s. Fixation inglutaraldehyde was done at 4°C for 2 h, followed by washingin cacodylate buffer (3 × 15 min), post-fixation in 1%m/vosmium tetroxide (OsO4) in cacodylate buffer in the dark for1 h (4°C) and washed again in MQ water (3 × 10 min).

After fixation, the specimens were sectioned into sixportions, dehydrated in a progressive series of ethanol(70–100% or 30–100% for glutaraldehyde-fixed samples)and embedded in paraffin (xylene was used for intermediateinfiltration). Some samples fixed in 2% glutaraldehyde v/v (in0.2M cacodylate buffer) and post-fixed in osmium tetroxidewere embedded in Epon 812 (Sigma-Aldrich, St. Louis, MO,USA), following Luft’s mixture. In brief: samples were dehy-drated up to 100% acetone, intermediately infiltrated withEpon+ 100% acetone (1:1) before final embedding andpolymerization at 60°C in gelatine capsules. Other samplesfixed and post-fixed similarly were embedded in London ResinWhite (LRW) (Fluka, Basel, Switzerland), using ethanolfor dehydration and ethanol + LRW (1:1) for intermediateinfiltration. Paraffin sections, 3–5 µm thick, were obtainedusing a Jung RM 2035 rotary microtome (Leica Microsystems,Wetzlar, Germany). Semi-thin (0.5–1 µm) and thin (≈90 nm)sections (Epon- or LRW-embedded samples) were obtainedwith a Reichert-Jung Ultracut E ultramicrotome (LeicaMicrosystems).

Histology and histochemistryMicrostructural and histochemical analyses were performedwith a tetrachrome technique combining Alcian Blue(AB), pH 2.5, for acidic sugars; Periodic Acid/Schiff’s (PAS)for neutral polysaccharides; Weigert’s iron Hematoxylinfor chromatin, and picric acid for muscle and cytoplasm wasemployed, following Costa & Costa (2012), with modifica-tions employed by Costa et al. (2014), for samples fixed inBouin’s, Carnoy’s or glutaraldehyde and embedded either inparaffin or Epon. In the latter case, the resin was partiallyremoved with Maxwell’s solution (KOH, saturated, in a2:1 methanol + propylene oxide mixture) for 5 min at roomtemperature to allow infiltration of the dyes. Other general

92 Ana P. Rodrigo et al.

staining procedures were applied to some sections for struc-tural detailing, such as Weigert’s Hematoxylin/van Gieson’smultichrome (Acid Fuchsin+ picric acid) and Toluidine Blue,the latter for Epon- and LRW-embedded samples only.

Lipid detection was achieved in samples post-fixed withosmium tetroxide and counterstained with Nuclear Fast Red.Protein staining was done using Coomassie Brilliant BlueR250 (Fisher, 1968). Other techniques include von Kossa’sreaction for calcium and Perl’s Prussian Blue for irondeposits. Bacteria detection was according to the protocol ofGram (1884), on paraffin-embedded sections.

Paraffin-embedded sections were cleared in xylene follo-wing staining. All slides were mounted with DPX resinousmedium. Standard histological and histochemical techniquescan be found, e.g., in Martoja & Martoja (1967) and Kiernan(2008). Optical microscopy analyses were done with a DMLBmodel microscope (Leica Microsystems).

Cytological analysesThin sections were collected onto copper mesh gridsand stained with 2% (m/v) aqueous Uranyl Acetate andReynold’s Lead Citrate. Analyses were done using a 1200EXmodel TEM (JEOL, Peabody, MA, USA) operated at 80 kV.

RESULTS

General anatomy of the digestive tractThe digestive system of E. viridis is formed by a strongmuscular eversible pharynx (proboscis), the esophagus, theintestine and rectum. Structures such as a crop or gizzard areabsent. The anterior portion of the digestive tract constitutesapproximately one-third of its length and comprises thepharynx and esophagus, both highly muscular (especially theformer), while the posterior section is chiefly constituted bythe intestine, followed by the rectum and anus. Betweenthe two sections the tract forms a loop to compensate thedistension of the proboscis (Fig. 1a). Sections through thefold revealed, as expected, the foregut (posterior pharynx and

esophagus) and the anterior portion of the intestine (Fig. 1b).The digestive process in E. viridis was observed to be con-tinuous, following the rate of ingestion of food items, whichvaries between undifferentiated material obtained frombarnacles, mussels and even other E. viridis.

PharynxThe proboscis is densely muscular and devoid of jaws orhook-like structures at its terminus, which is accordant withits suction functions. The worm was observed to be able touse this structure to penetrate between the valves of livemussels. The organ was also seemingly used to sense theenvironment. Lining the pharynx toward the exterior, thedense circular musculature of the pharynx was overlaidby longitudinal muscular fascicles between which nerve bun-dles were found. The proboscis was covered externally by anepithelium containing many pigment cells and AB-positive(blue) glandular cells, thus revealing secretion of acidicmucopolysaccharides. To this epithelium were attached manysensory papillae (Fig. 2a). The terminus of the proboscis con-tained longer, palp-like papillae with many mucous-secretingglandular cells (Fig. 2b). The inner epithelium of the pharynxformed small villi, being pseudo-stratified and covered by acuticle and an epicuticle. The epithelium was comprised ofpigment and serous glandular cells. The serous cells produceddensely packed secretory vesicles that should contain theinactive precursors of digestive enzymes (forming zymogengranules), whenever food items were observed passing throughthe foregut. These cells were found to be devoid of significantamounts of secretory vesicles when food was undergoingprocessing in the intestine (Fig. 2c).

EsophagusThis component of the foregut was observed to be muchshorter than the pharynx but still heavily muscular. Thestructure of the villi in the esophagus was found to be morecomplex than in the pharynx, the villi being conspicuouslylonger. The inner epithelium also had a well-defined cuticle

Figure 1. The fold in the gut of Eulalia viridis, linking the foregut with the intestine. a: Macroscopic aspect of the foldrevealed by a simple longitudinal incision through the animal, revealing the anatomical connection between the pharynx(ph), esophagus (oe) and intestine (int). b: Histological section across the fold (tetrachrome stain, specimen fixed inCarnoy’s), showing the posterior end of the pharynx (ph), the esophagus (oe) and the anterior most part of the intestineconnected with the esophagus (oe/int). Parapodium (pd); ventral nerve cord (vn). Scale bar: (a) 1 mm, (b) 400 µm.

Microstructure of the Gut of a Marine Annelid 93

and epicuticle, as the pharynx, albeit a thicker layer ofPAS-reactive fibers underlying the epithelium (Fig. 3).Serous glandular cells, similar to those in the pharyngealepithelium, were also found in the esophagus. These cellsunderwent similar changes along the digestive process asthose mentioned above (Fig. 3—inset).

IntestineThe intestine was formed by a thin muscular layer overlaidby a columnar-like epithelium with variable thickness chieflyformed by a single layer of cells (Fig. 4a). The epithelium

formed villi-like structures whose size and shape changedaccording to the digestion phase. Digestive cells were themost common and were characterized for being long (albeitvariable in size), holdingmany irregularly sized inclusions andvacuoles and bearing apical microvilli. The nuclei of digestivecells were found closer to the epithelial basal membrane.A set of cells, likely holding secretory functions, were clearlydistinguishable from digestive cells due to their dense cyto-plasm and the presence of spherical vacuoles of hyalineappearance (Fig. 4b). The contents of the vacuoles werereleased to the lumen of the gut through exocytosis. Twoseemingly distinct types of secretory cells were observed,as shown by distinct histochemical properties of vacuoles(see below). At an earlier stage of digestion, most secretory cellsheld vacuoles that had a hyaline yellow color following stainingwith picric acid. Besides the aforementioned secretory anddigestive cells, basal cells could occasionally be found com-pressed against the basal membrane of the epithelium. Thesesmall cells, likely involved in epithelial regeneration, were rareand detected best in resin-embedded samples (Fig. 4b).

Whereas the three cell types were present in the epitheliumregardless of digestive phase, the digestive cells showed themost significant changes along the process. Although aclear separation of phases could not be pinpointed, due to thecomplexity and continuity of the digestive process, threemain phases were distinguishable: secretory, absorptive andexcretory. The secretory phase consisted of the earliest phase ofthe digestive process, being characterized by the production andsecretion of AB-positive substances (therefore acidic mucopo-lysaccharides) allocated within large vacuole-like structures,forming “blue bodies” within digestive cells (Fig. 5a). Thedigestive cells failed to reveal significant accumulation ofPAS-positive substances (i.e., glycogen-like sugars) or digestivevacuoles, however, numerous vesicles were produced, likely

Figure 2. Sections across the proboscis (tetrachrome stain). a: Transversal section of the eversible pharynx, i.e., theproboscis (ph), showing the pharyngeal musculature (phm) stained yellow by Picric acid, as well as the outer transversalmuscles (tm). Between tm, nerve cords (nc) are fibrous in appearance and tend to be stained by both Alcian Blue (AB;blue) and Periodic Acid/Schiff’s (PAS; pink). Sensory papilla (sp), also reactive for AB and PAS, surround the pro-boscis. Specimen fixed in glutaraldehyde. b: Longitudinal section of terminal sensorial papilla with AB-reactive muco-substances (ms) plus serous cells (sc) and respective nuclei (n). Specimen fixed in glutaraldehyde. c: Cross section of theinner wall of the pharynx, revealing the dense pharyngeal musculature containing longitudinal and transversal fibers(phm), serous cells (sc) and nerve cells (nv). Pigment cells (pc) containing vesicles of greenish substances are foundcompressed between serous cells. The cuticle (ct) is PAS-positive (pink) while the epicuticle (ect) tends to beAB-positive (blue). Specimen fixed in Bouin’s. Scale bar: (a) 300 µm, (b) 50 µm, (c) 25 µm.

Figure 3. Section across the esophagus at resting phase, revealingserous cells (sc) with few vesicles. Note the strong pink colorationof the cuticle (ct). Nuclei (n); musculature of the esophagus (oem).Inset: Serous cell (sc) with numerous secretory in a moreadvanced stage of digestion. Tetrachrome stain (specimen fixed inCarnoy’s). Scale bar is 50 µm.


containing digestive enzymes. The initial stage of digestion washighlighted when undigested food items were still found in theintestinal lumen (Fig. 5b). No significant signs of bacterialcolonies were found at this or any other stage of digestion.

During the absorptive phase, digestive cells accumulatedagglomerates of glycogen-like substances (PAS-positive,stained bright pink) while mucosubstances became scarcer(Fig. 6a). The secretory cells were also observed to initiatefunction as seen by an increase in number of vacuoles.Furthermore, the existence of two types of these cells becamemore obvious. The contents of the secretory vacuoles weremostly stained by picric acid and, in some cases, PAS.Typically, the contents of the vacuoles were homogenous,amorphous and electron-dense. However, some vacuolesheld a membrane that was boldly stained by AB, providingan overall “bluish” tint. The two types of vacuoles were notpresent in the same cells, which were hitherto divided into

cells possessing “yellow” and “blue” secretory vacuoles(Figs. 6a, 6b). Secretory cells possessed a denser cytoplasmthan digestive cells (Fig. 6b—inset). The contents of “blue”vacuoles were also more intensely stained by PAS andToluidine Blue (Fig. 6c). The membrane of “blue” vacuoleswas also strongly stained by Coomassie Blue (not shown),indicating, together with AB, the existence of undisclosedacidic glycoproteins. Still, the Coomassie blue staining ofthe contents of the vacuoles was relatively dim comparedwith their membrane. Besides glycogen-like substances, thedigestive cells also gather many other diffuse inclusions,from pigmented granules to vesicular structures that tendedto aggregate around nuclei, most of which should containzymogen-like substances (Fig. 6d). Among these inclusions,small spherical structures consistent with organometallicexcretory structures such as sphaerocrystals Saulnier-Michel,1992) were occasionally found (not shown) in TEM

Figure 4. Transversal sections in the intestine of a worm fixed in glutaraldehyde and embedded in Epon. a: Generaloverview of the intestinal epithelium (ep) and adjacent muscle layer (ml). Basal membrane (bm); body wall musculature(bw); coelom (co); gut lumen (lm); oocyte (oo); villi (vl) (tetrachrome stain). b: Section across the intestinal epithelium,revealing distinct cell types in the epithelium; digestive (dc), secretory (sc) and basal (bc) cells. Basal membrane (bm);gut lumen (lm); muscle layer (ml); nuclei (n) (Toluidine Blue stain). Scale bar: (a) 50 µm, (b) 25 µm.

Figure 5. Section through the intestine during early (secretory) digestion stage (Tetrachrome stain on paraffin-embedded specimens fixed in Carnoy’s or Bouin’s). a: Section through epithelium during the secretory phase, contain-ing mostly Alcian Blue (AB)-positive (indicating mucosubstances) “blue bodies” (bb) and many digestive vesicles(dv) inside digestive cells. Developing secretory cell (sc). Inset: details of “blue bodies”. b: Fairly undigested prey Eulaliaviridis inside the gut of a larger worm (py). The intestinal epithelium (ep) is seemingly at the same stage as in thepreceding panel. Note the intact gut of the prey (*). Inset: another E. viridis turned prey, although at a more advancedstage of digestion. Scale bar: (a) 25 µm, (b) 300 µm.


preparations from the epithelia of sections at a moreadvanced stage of digestion.

The excretory phase of digestion involved the formationof apical corpuscles primarily in the mid and posteriorsection of the intestine, indicating the release of undigestedmaterials and digestive by-products (Fig. 7a). These struc-tures were observed to be shed to the lumen of the gutand did not consist of full cells, rather portions of digestivecells containing amorphous materials (Fig. 7b). Secretorycells (both types) were still present at this stage. The cyto-plasm of digestive cells hitherto held many agglomerates ofPAS-positive substances, with a tendency to increase in sizerelatively to the previous stage (Fig. 7c). The number, size,and variety of endoplasmatic inclusions in digestive cellsincreased at this stage, with particular respect to pigment-bearing vesicles and corpuscles, which ranged in color fromgreen to brownish-orange in unstained slides (Fig. 7d).

Few calcium deposits (from von Kossa’s reaction onparaffin-embedded sections) were found in the intestinalepithelium. However, these became more evident in a latterphase of digestion, typically around digestive vacuoles,

in relation to the presence of sphaerocrystals mentionedabove (not shown). Iron deposits were not found in the gutepithelia, regardless of digestive phase. In paraffin- andresin-embedded samples subjected to osmium tetroxidepost-fixation, small lipid droplets (stained blackish) couldbe found in digestive cells, also in a latter digestion phase(Fig. 7a—insets). In general, lipids were scarce in E. viridisdigestive tract and always limited to small droplets.

RectumThe rectum is within the narrow final segments of the worm,which were often absent and healing from pre-collectioninjury. The epithelium differs considerably from the remainingcomponents of the gut, including the intestine (Fig. 8). Theepithelium was relatively thin, strongly reactive to PAS andhighly basophilic (high affinity to Hematoxylin), indicating amore acidic intracellularmedium (thus rendering a dark tint tocells). As previous, the epithelium was comprised of a singlecell layer and markedly pseudostratified. Only a single cell typecould be clearly differentiated in the epithelium of the rectum.,

Figure 6. Micrographs of the intestinal epithelium during the absorptive phase. a: Digestive cells contain many irregularagglomerates of Periodic Acid/Schiff’s (PAS)-positive (bright pink) glycogen-like substances (ga). Secretory cell (sc); nuclei(n). Tetrachrome stain on paraffin-embedded section (glutaraldehyde fixation, tetrachrome stain). b: Epon section ofepithelium (tetrachrome stain) evidencing secretory cells containing “yellow” (yv) and “blue” (bv) vacuoles. The digestivevacuoles (dv) are heterogeneous in shape, size and contents. Glycogen agglomerates (ga); gut lumen (lm); nuclei (n). Inset:transmission electron microscopy (TEM) micrograph of the apical part of digestive (dc) and secretory cells (sc). Note themicrovilli (mv) border and the electron-dense secretory vacuoles. c: Toluidine Blue-stained Epon section of the epithelium(grayscale) revealing distinct metachromasia between the two types of secretory cells. Blue secretory vacuoles (bv); yellowsecretory vacuoles (yv). Note the exocytosis process (ex). Digestive vacuoles (dv). d: TEM micrograph of digestive cellnuclei (n), evidencing clear nucleoli (nc); euchromatin (eh) and heterochromatin (hh). Vesicles of undisclosed substances(sb), likely digestion products, revealed here to hold low electron density. Scale bar: (a, b, c) 25 µm, (d) 2.5 µm.


Its function could not be entirely disclosed, although it islikely involved in absorption and storage. There is no definiteborder between the rectum and the intestine. The cytoplasmis dense and granular in appearance due to the presenceof numerous vesicular structures containing PAS-reactivesubstances that could not be identified histochemically. Occa-sionally, intraplasmatic bodies heavily stained by Weigert’sHematoxylin (blackish) and green pigments were visible. It islikely that these dark bodies containing amorphous materialare particulate waste from the digestive process. Clumps of thismaterial were also found between the intestine and the bodywall similar to observations from other species of polychaetes(e.g., Marsden, 1968). No significant microanatomical orgeneral morphological changes were found to occur along thedigestive cycle.

DISCUSSION

The present study showed that, albeit anatomically simple, themarine polychaete E. viridis exhibits complexmicroanatomical

Figure 7. Sections across the intestinal epithelium during the excretory phase. a: Advanced digestion phase where theepithelium (tetrachrome stain on paraffin sections) glycogen-containing bodies (ga) are larger and more dense than inthe absorptive phase. Note the apical corpuscles (ac) being released into the lumen (lm). Insets: lipidic droplets (arrows)evidenced by osmium- tetroxide in paraffin-embedded samples, counterstained with Nuclear Fast Red (upper right);and in London Resin White (LRW)-embedded samples, viewed by transmission electron microscopy (TEM; lower left).b: Epon section (Toluidine Blue stain), showing glycogen agglomerates (ga), the release of apical corpuscles (ac) intothe lumen (lm) and the two types of secretory cells, i.e., containing “blue” and “yellow” vacuoles (bv and yv, respec-tively). c: Advanced excretory phase, tetrachrome stain (paraffin section), where the epithelium contains many PeriodicAcid/Schiff’s (PAS)-positive (pink) structures, likely glycogen agglomerates (ga), secretory vacuoles, mostly bv, andapical corpuscles (ac). d: Unstained epithelium (paraffin section, glutaraldehyde-fixed sample), showing naturallycolored greenish pigments (gp) held in microvesicles located diffusively, although more densely near the gut lumen(lm) and brownish/orange-pigmented agglomerates (pa). Scale bar is 25 µm.

Figure 8. Longitudinal paraffin section of posterior segments ofEulalia viridis (glutaraldehyde fixation, tetrachrome stain). Notethe thin, heavily stained rectum epithelium (rt), owing to Wei-gert’s Hematoxylin and Periodic Acid/Schiff’s (PAS) reaction, andthe blackish agglomerates of undifferentiated material (ba). Nuclei(n); parapodia (pd). Scale bar is 25 µm.


changes in the gut epithelium during the digestive process thatultimately reflect the cumulative functions of this organ. Theobservations show that the cells of the digestive epitheliumundergo several phases that relate to the steps of the digestiveprocess: resting/secretory (early phase of digestion), digestive(absorptive) and excretory (final stage of digestion). Thesedifferent steps are reflected in alterations of cellular morphol-ogy and function, as inferred histologically and histochemi-cally. However, the different stages of digestion do not appearto imply the differentiation and subsequent replacement ofnew digestive cells to new functions, but rather a progressiveshift in the physiology of each individual cell. In fact, nosignificant shedding of whole epithelial cells was observed inany of the stages. The cell types and functions varied withcomponent of the digestive tract, but not with digestive stage,although cells, especially those of the intestine, underwentvery significant changes throughout the process. Importantly,two types of secretory cells were identified in the intestinalepithelium, one bearing vacuoles holding substances withhigher affinity to picric acid, another with vacuoles enclosingseemingly more PAS-reactive substances and characterizedby a dense, glycoprotein-rich membrane. These cells will hereforth be referred to as type “A” and “B”, respectively. Theexistence of two types of secretory cells was disclosed byMichel(1969b), hitherto referred to as glandular cells “type 1 and 2”,for which no absolute correspondence could be establishedbetween A and B cells herewith described. The same authorinferred proteolytic activity of the substances inside thevacuoles from indol and disulphide bonds, which should implya function related to secretion of digestive enzymes. Still,no specific enzymes could be linked to either type, as in thepresent study. Nonetheless, the specific function of either typeduring the digestive cycle was not investigated by Michel(1969b), who did not focus on different stages of the digestivecycle., The time-lapse between the peaks of activity of the twotypes of cells suggests distinct physiological roles along thedigestive cycle.

Eulalia viridis has the basic anatomical components ofthe polychaete digestive tract, encompassing an eversiblepharynx (proboscis), followed by the esophagus, an uncoiledintestine devoid of specialized structures, like caeca, and therectum. There is no stomach which is a feature common insedentary polychaetes (see Saulnier-Michel, 1992). Table 1summarizes the main components of E. viridis digestivetract. Previous research disclosed that components of thepolychaete digestive tract may be divided in more or lessspecialized regions, especially the intestine (see, e.g., Pilgrim,1965). However, such subdivision was not found in E. viridis,which may relate to the opportunistic feeding habits of thespecies that render the gut apt for processing a wide varietyof prey, which is in agreement with reduced or absentspecialization of external feeding-related structures (such asjaws or palps) other than the powerful proboscis that is fittedwith many small sensory papillae. This is in accordance withthe presence of multiple nerve bundles underneath the skinthat connect to the papillae (Fig. 2) in agreement withobservations by Michel (1964). In fact, the current findingsindicate that the proboscis may be one of the most importantsensory organs of the worm, which agrees with personalobservations of E. viridis using the proboscis to sensetheir environment when scouting for food in mussel beds,and potentially through chemical sensing (Michel, 1970).Absence of a stomach, as seen in E. viridis, was reportedpreviously for polychaetes (Welsch & Storch, 1970) andespecially in Phyllodocidae, albeit hitherto not for E. viridis(Tzetlin & Purschke, 2005). The stomach of polychaetespecies, when present, is usually regarded as the mainorgan of digestion, varying between more markedly gland-ular or more mechanical (muscular) functions (Sutton,1957; Pilgrim, 1965). In E. viridis, the dense musculature ofthe pharynx, together with its densely packed glandular(secretory) epithelial cells, overtakes the role of the stomach,leaving to the intestine the bulk of the intracellular bio-chemical processes.

Table 1. Cell Types and Respective Function in the Gut Epithelia of Eulalia viridis.

Organ Cell Type Cell Function

Pharynx Mucous cells Mucous secretionSerous cells Enzyme secretionPigment cells Pigmentation

Esophagus Serous cells Enzyme secretionIntestine Basal cells Epithelium renewal

Digestive cells Mucous secretionEnzyme secretionIntracellular digestionStorage (mostly sugars)Excretion (salts, digestive by-products)

Type “A” secretory cells Enzyme secretionType “B” secretory cells Enzyme secretion

Rectum Single-type epithelial cells AbsorptionStorageElimination of particulate digestive waste


The only study reporting microanatomical changesduring digestion in E. viridis was performed by Michel(1969a), on a specific organ, the pharynx, and comparedfasting worms with worms immediately fixed after feedingon a dead mussel. This study focused mostly on changes inenzyme-secreting cells. In accordance with this study, thedigestive epithelium of the pharynx is chiefly constituted byglandular (serous) cells specialized in secretion of digestiveenzymes to the gut lumen. These cells become more activeduring digestion, which has been revealed by the low density ofgranular inclusions (likely zymogen granules) within thesecells after food items have been processed through this part ofthe alimentary canal (Fig. 2c). This is consistent with theobservations of Michel (1969a), who reported loss of secretoryvesicles from serous cells following ingestion. Michel (1968)also disclosed that most of the enzymes secreted by serous cellsare proteolytic, concluding that this is consistent with predatoryfeeding habits. Also agreeing with the work performed byMichel (1969a), a thick bi-layered cuticle lining the epitheliaof the pharynx and the esophagus is present. The cuticle(composed mostly of polysaccharides, should protect theepithelium from autolysis while impeding intracellulardigestion, a function that is clearly limited to the intestinalepithelium. The fact that no significant changes in themicroanatomy of the pharynx epithelium were noticed, apartfrom depletion of secretory vesicles from serous cells, alsocorroborates this. The esophagus constitutes a relativelyshort section of the digestive tract that shares many char-acteristics with the pharynx with respect to cell type andfunction as a hinge between the pharynx and the intestine.As in the present work, other authors also mentioned thepresence of secreting cells in the esophagus (e.g., Tzetlinet al., 1992), but the precise biochemical nature of thesecretions needs to be disclosed.

The intestine of E. viridis had the most significant chan-ges according to progression of the digestion phases. Theepithelial cells of E. viridis intestine combined the functions ofdigestion, metabolization/excretion and storage. In absenceof detailed information for invertebrate digestive epithelia,polychaetes in particular, comparisons may be drawn fromthe molluscan digestive gland, especially concerning cepha-lopods, for which there is sizable literature reportingthe multiple functions of epithelial cells, including enzymesecretion, absorption and excretion (Boucaud-Camou & Yim,1980; Swift et al., 2005; Costa et al., 2014).

The digestive cells of E. viridis are by far the mostnumerous and undergo very significant alterations duringthe digestive process. These cells, in early stages of digestion(Fig. 5), are mostly dedicated to mucous secretion (of acidic,non-sulphated, mucopolysaccharides, AB positive), which isfollowed by active secretion of enzymes, that are arrangedinto densely packed vesicles likely containing zymogen(secretory phase). Although microstructural variations dur-ing the digestive process were hitherto not surveyed, Michel(1969b) found that enzymes secreted by intestinal epithelialcells are mostly proteases. Conversely, other authorsreported the existence of specialized mucous-secreting cells

in the intestine of polychaete worms (e.g., Pilgrim, 1965;Saulnier-Michel et al., 1990). Still, whether other polychaetespossess specialized mucous-secreting cells in the intestinalepithelia or these are, in fact, digestive cells in earlier stagesof digestion lacks confirmation. Additionally, it is possiblethat the uneven distribution of mucosubstances along theintestine of some polychaete annelids described by otherauthors actually results from different stages of the digestiveprocess (Sutton, 1957; Michel, 1977).

The absorptive phase is chiefly characterized by thepresence of mildly PAS-positive bodies and digestivevacuoles (Saulnier-Michel et al., 1990). During this stage,glycogen-like material progressively turns into PAS-positiveaggregates, usually located at the apical portion of the cells(Fig. 6). The digestive cells have a dense microvilli brushborder facing the lumen of the gut, as in several polychaetespecies (Welsch & Storch, 1970). At the excretory phase,the digestive cells release to the lumen of the gut digestiveby-products, undigested material and perhaps even excre-tory substances (like calcium salts in sphaerocrystals) in theform of apical corpuscles (Fig. 7). The apical corpusclesshare some similarities with the “brown bodies” describedfor cephalopods, which are membrane-bound structurescontaining by-products of the digestive process that arereleased to the lumen of digestive diverticula at moreadvanced stages of digestion (Costa et al., 2014).

A role for digestive epithelia in excretion has long beensuggested for polychaete annelids, such as the stomach of thehydrothermal vent wormAlvinella pompejana (Saulnier-Michelet al., 1990) or the intestine of the bristleworm Hermodicecarunculata (Marsden, 1968). Still, there is little information onthese animals, unlikemolluscs, amongmarine invertebrates. ForA. pompejana, it has been suggested that digestive cells activelyproduce sphaerocrystals to eliminate metals (the worm inhabitsa strongly toxic environment); in H. carunculata intestinalexcretion has been disclosed as a route of elimination of non-particulate, homogeneous, materials without, however, micro-anatomical detailing of the process. The same work alsomentions the hindgut epithelium is involved in elimination ofsolid, amorphous, digestive waste, which is in accordance withthe current observations of the rectal epithelia (Fig. 8). Sincesphaerocrystals or significant deposits of metals, besidescalcium during the excretory phase, were scarce in the epi-thelia of the worm, it may indicate species-specific excretoryprocesses for E. viridis related to its feeding and habitat.Overall, the study of intestinal excretion in annelids shouldbenefit from further research.

Only scant lipid reserves were found in the gut ofE. viridis, in the form of minute lipid droplets scattered indigestive cells at later stages of the digestive process (Fig. 7a).In fact, the main energy reserve of the species appears to beglycogen (Figs. 6, 7). Michel (1969b) reported much reducedlipase activity, compared with that of proteolytic enzymes, inthe alimentary canal of this species, which aids explainingthe reduced lipid deposits within digestive cells. There is littleinformation on lipid absorption and storage in polychaetespecies. However, some authors reported the abundance of


lipid droplets in epithelial cells of the gut in some sedentaryworms (Pilgrim, 1965). Other authors describe highly vari-able interspecific accumulation of lipids and glycogenin Errantia (Welsch & Storch, 1970). In a hydrothermalphyllodocid (Galapagomystides aristata) many minute lipiddroplets were found in the epithelial cells of the midgut(Jenkins et al., 2002), which is in better agreement with thecurrent findings. In face of the high variability regarding themain reserve substances (i.e., fat versus sugar) it may beinferred that there is no clear trend among polychaetes,regardless of habitat and behavior. Thus, the absorption,metabolism and storage of typical reserve substances in theseanimals are highly dependent on undisclosed physiologicaltraits and energy expenditure. Interestingly, the organismsbest related to polychaete species with respect to similaritiesbetween the cells of digestive epithelia, the cephalopodmolluscs, are known to store very significant amounts oflipids in epithelial cells of the digestive gland, at least in moreadvanced stages of digestion (e.g., Boucaud-Camou & Yim,1980; Moltschaniwskyj & Johnston, 2006; Costa et al., 2014).

Finally, it must be noted that pigment-containing vesiclesor agglomerates were found throughout the intestinal epithelia(Fig. 7). Whereas some of these pigment agglomerates may besimilar to zymogen granules, the source or purpose of thegreenish pigments cannot, at this stage, be identified. However,it is possible that E. viridis owes it bright-green integumentarycolor to pigments that are absorbed and/or metabolized indigestive epithelia, quite similar to what has been observedin the sedentary polychaete Chaeopterus spp. (Kennedy &Nicol, 1959).

CONCLUSION

Organisms with reduced organ differentiation and speciali-zation are prone to have complex sub-cellular physiologicalprocesses that reflect the accumulation of functions withina given type of cell. In E. viridis, the most obvious arepolyvalent digestive cells of the intestine. These cells functionin secretion of enzymes and mucosubstances, intracellulardigestion, storage and by-product transformation and elim-ination. Furthermore, these cells undergo microanatomicaland physiological changes during the digestive process, withno significant replacement of cells being observed during thecontinuous processing of food items. The secretory functionsof the intestinal epithelium are complemented by theexistence of two types of specialized secretory cells that,albeit morphologically similar, are likely responsible for theproduction distinct substances involved in the digestiveprocess (such as proteolytic enzymes). Altogether, the find-ings disclosed a relatively simple anatomy of the digestivetract in accordance with the notion that E. viridis feeds on awide variety of food items. Hence, the adaptative value of nothaving over-specialized structures related to feeding anddigestion, other than the highly muscular and sensorialeversible pharynx. The present study also highlights theexistence of three distinct, albeit interlinked, phases in thedigestive process of this annelid and showed that the lack of

knowledge of the physiological and microanatomical chan-ges during the digestion of polychaete species results inconfusion regarding the identification of cell types and theirfunctions.

ACKNOWLEDGMENTS

The authors acknowledge Fundação para a Ciência eTecnologia for the grant SFRH/BPD/72564/2010 to P.M.Costa and for funding the project EXPL/MAR-EST/1236/2012, which includes the fellowship to A. Rodrigo. Theauthors also acknowledge the contributions of C. Gonçalves,C. Martins and J. Santos (IMAR); P. Henriques (CiiEM) andL. Saramago (FCUL).

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