Microscopical study of the crop and oesophagus of the carnivorous opisthobranch Philinopsis depicta (Cephalaspidea, Aglajidae)

MICROSCOPICAL STUDY OF THE CROP AND OESOPHAGUS OF

THE CARNIVOROUS OPISTHOBRANCH PHILINOPSIS DEPICTA

(CEPHALASPIDEA: AGLAJIDAE)

ALEXANDRE LOBO-DA-CUNHA1,2,3, TANIA SANTOS2, ELSAOLIVEIRA1,ANGELA ALVES1, RITA COELHO3 AND GONCALO CALADO3,4

1Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, Portugal;2Centre of Marine and Environmental Research (CIIMAR), 4050-123 Porto, Portugal;

3Portuguese Institute of Malacology (IPM), 8201-864 Guia, Portugal; and4Marine and Environmental Research Centre (IMAR), FCT/UNL, 2829-516 Caparica, Portugal

Correspondence: A. Lobo-da-Cunha; e-mail: [email protected]

(Received 22 November 2010; accepted 10 June 2011)

ABSTRACT

A histochemical and ultrastructural study of the crop and oesophagus was carried out for the firsttime in Aglajidae. In Philinopsis depicta, the hind region of the crop contains two large folds creating achannel between them, lined by a ciliated epithelium. This ciliated groove continues through the pos-terior oesophagus. In addition to these larger folds, both crop and posterior oesophagus presentmany smaller longitudinal ridges lined by a nonciliated epithelium formed by cells bearing microvilliembedded in a layer of extracellular material. Lysosomes and mitochondria are common in thesupranuclear region of these cells, and in the basal region hemidesmosomes are frequent. Epithelialsecretory cells contain many large vesicles with a low electron-density content rich in acid polysac-charides, but without detectable amounts of protein. The basal region of the secretory cells comprisesthe nucleus and several Golgi stacks formed by many flat cisternae with low electron-density content.Secretory cells were not observed in the ciliated epithelium of the channel between the two largefolds. The connective tissue of the crop and posterior oesophagus contains many large vacuolar cellswith a thin layer of cytoplasm around the single vacuole that occupies about 90% or more of the cellvolume. Tubular invaginations of the cell membrane are frequent in these cells and some alsocontain large vesicles that can open to the central vacuole, suggesting an intense transport activityacross the cytoplasm. These vacuolar cells could be related to the calcium cells reported in the con-nective tissue of other gastropods. Ultrastructural and histochemical studies of the organs of the diges-tive system in carnivorous and herbivorous cephalaspideans can lead to the discovery of importantunknown features, like the vacuolar cells, which might be useful to establish correlations withmolecular phylogenetic data or food sources.

INTRODUCTION

Aglajids are known as active predators, feeding mainly onother opisthobranchs, polychaetes, nemerteans and flatworms,although the prey of several species has not yet been reported(Gosliner, 1980; Rudman & Willan, 1998). Aglajids possess abulbous or tubular muscular buccal bulb that can be eversiblein some species (Gosliner, 1980; Rudman & Willan, 1998).The prey is ingested by the suction generated by the muscularbuccal bulb or grasped by the everted buccal bulb anddigested within the crop. A digestive fluid containing enzymessecreted by the digestive gland reaches the crop where the softtissues of the prey are dissolved. Afterwards, a nutrient-rich

fluid proceeds to the stomach, which is embedded in the diges-tive gland and linked to it by ducts. Endocytosis, intracellulardigestion and nutrient absorption take place in the digestivegland and the undigested parts of the prey, including shells,are defaecated or regurgitated (Rudman, 1972b). Nonetheless,extracellular digestion followed by endocytosis and intracellulardigestion in lysosomes, nutrient uptake and accumulation ofglycogen granules and lipid droplets can also occur in the oeso-phagus and crop of gastropods (Walker, 1972; Bourne, Jones &Bowen, 1991; Lobo-da-Cunha & Batista-Pinto, 2005;Lobo-da-Cunha et al., 2010a).Although absent in aglajids (Rudman, 1974; Gosliner,

1980), a muscular gizzard with three plates is considered a

# The Author 2011. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved

Journal of The Malacological Society of London

Molluscan StudiesJournal of Molluscan Studies (2011) 77: 322–331. doi:10.1093/mollus/eyr031Advance Access publication date: 19 September 2011

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synapomorphy of the cephalaspideans (Mikkelsen, 1996).However, there is no correlation between the presence of agizzard and the trophic position, because this structure can bepresent or absent in either carnivorous or herbivorous cepha-laspideans (Malaquias, Bericibar & Reid, 2009).

The Aglajidae are one of the families of cephalaspideans inwhich all species have a carnivorous diet, but other cephalaspi-dean families contain only herbivores (Kohn, 1983; Malaquiaset al., 2009). According to some studies, herbivory is the plesio-morphic condition in cephalaspideans, while carnivory seemsto have arisen independently two or more times during theevolution of this clade (Mikkelsen, 1996; Malaquias et al.,2009). Available data suggest that dietary specialization hasplayed a major role in the adaptive radiation of cephalaspi-deans, allowing a variety of feeding niches to be occupied(Malaquias et al., 2009). Still, adaptation to a new diet mustrequire morphological and functional modifications of thedigestive system that have not yet been thoroughly investi-gated. Among cephalaspideans, morphology of radula andgizzard plates have been analysed in great detail (Gosliner,1994; Mikkelsen, 1996; Malaquias & Reid, 2008); however,other parts of the digestive system have received less attentionin recent years. Some light-microscopic studies dedicated tothe digestive system of cephalaspideans were published manyyears ago (Fretter, 1939; Rudman, 1971, 1972a, b), but so farultrastructural studies are still scarce (Lobo-da-Cunha &Calado, 2008; Lobo-da-Cunha et al., 2010a, b).

Most studies on aglajids deal with general anatomy, taxon-omy, reproduction or ecology (Rudman, 1972c, 1974; Gosliner,1980; Anthes & Michiels, 2007). An article by Rudman(1972b) was specifically devoted to morphological and func-tional aspects of the digestive system of two aglajid species,Melanochlamys cylindrica and Philinopsis taronga, and a histochem-ical and ultrastructural study of the salivary glands of P. depictawas published a couple of years ago (Lobo-da-Cunha et al.,2009). Nevertheless, further studies are necessary for a detailedcomparison between the digestive system of carnivorous andherbivorous cephalaspideans.

The crop is an organ of major importance in the digestivesystem of aglajids, because extracellular digestion of prey takesplace in it, functioning like a stomach. On the other hand, theactual stomach is just a segment of the digestive tract wherethe ducts of the digestive gland open (Rudman, 1972b). Thus,to extend the current knowledge about the digestive system ofaglajids the crop should be investigated in detail. It was there-fore studied in P. depicta with both light and electronmicroscopy.

MATERIAL AND METHODS

Morphology

Six specimens of Philinopsis depicta (Renier, 1807) 7–8 cm inlength were collected in Ria de Alvor, an estuary on the southcoast of Portugal. For light microscopy the crop and posterioroesophagus of two animals were fixed in Bouin’s solution for24 h, dehydrated with ethanol and embedded in paraffin.Tissue sections were stained with haematoxylin and eosin, orwith Masson’s trichrome. Small fragments of these organs col-lected from the remaining four specimens were fixed for 2 h at48C in fixative containing 2.5% glutaraldehyde and 4% for-maldehyde (obtained from hydrolysis of paraformaldehyde)diluted with 0.4 M cacodylate buffer, pH 7.4 (final buffer con-centration 0.28 M). After washing in buffer, the fragmentswere postfixed with 2% OsO4 buffered with cacodylate, dehy-drated in ethanol and embedded in Epon. Semithin sectionsfor light microscopy were stained with methylene blue andazure II. Ultrathin sections for transmission electron

microscopy were stained with uranyl acetate and lead citrate,before being observed in a JEOL 100CXII transmission elec-tron microscope operated at 60 kV.

Histochemistry and cytochemistry

The tetrazonium coupling reaction for protein detection, peri-odic acid Schiff (PAS) reaction for polysaccharides and alcianblue staining for acid polysaccharide detection were applied todeparaffinized sections of crop and posterior oesophagus fixedwith Bouin’s solution (Ganter & Jolles, 1970).

Tetrazonium coupling reaction: sections were treated for10 min with a freshly prepared 0.2% solution of fast blue salt Bin veronal-acetate buffer, pH 9.2, washed and treated for15 min with a saturated solution of b-naphthol inveronal-acetate buffer, pH 9.2.

PAS: sections were treated with 1% periodic acid for10 min, washed and stained with Schiff’s reagent for 15 min.In control sections the oxidation with periodic acid wasomitted.

Alcian blue: sections were stained with a 0.5% solution ofalcian blue diluted in 3% acetic acid of pH 2.5 for detection ofcarboxylated polysaccharides and in an HCl solution of pH1.0 for detection of sulphated polysaccharides.

Sections were stained with haematoxylin, dehydrated andmounted with Entellan medium.

For ultrastructural detection of polysaccharides, ultrathinsections were treated with a 5% solution of tannic acid for10 min, briefly washed in water, stained with 3% uranylacetate for 10 min and finally washed in water (Sannes,Katsuyama & Spicer, 1978).

RESULTS

Anatomy, histology and histochemistry

The digestive tract of Philinopsis depicta includes a large muscu-lar buccal bulb connected to a voluminous crop (Fig. 1A). Thecrop of one specimen contained a large shell of Bulla striata thatwas perfectly clean with no traces of soft tissues. This shell wasof practically the same size and shape as the buccal bulb andcaused a substantial distension of the crop wall, which becamethin and translucent over a large part of its surface (Fig. 1A).Especially when the crop is completely filled, an anterior oeso-phagus between the buccal bulb and the crop is practicallyundetectable, but the posterior oesophagus is clearly recog-nized (Fig. 1A). The crop of P. depicta contains two large longi-tudinally oriented folds, reaching a height of c. 3 mm inhistological sections. A channel is formed between these folds,extending from the middle of the crop to the posterior oesopha-gus, and the free edge of the folds is curved towards theinterior of the channel between them (Fig. 1B, C).

The crop wall forms many longitudinal ridges, about 1 mmhigh or less in histological sections, which can also be recog-nized on the external surface of the two large folds (Fig. 1C).These ridges are lined by a nonciliated epithelium covered bya thin layer of extracellular material that stains with haema-toxylin (Fig. 1D). Large secretory cells are inserted in the non-ciliated epithelium, being weakly stained by haematoxylin(Fig. 1D). The epithelium covering the outer surface of thelarge folds is equal to the nonciliated epithelium lining theridges of the crop wall in general and also contains secretorycells, but secretory cells were not observed in the ciliated epi-thelium lining the channel between the two large folds of thecrop posterior zone (Fig. 1E).

The histochemical analysis reveals that the secretory cells arerich in acid polysaccharides, but without detectable amountsof protein. These cells are strongly stained by PAS reaction

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and alcian blue at pH 2.5 (Fig. 1F, G), being weakly stainedby alcian blue at pH 1 (Fig. 1H) and unstained by the tetrazo-nium coupling reaction for protein detection (Fig. 1I). Thelayer of extracellular material covering the nonciliated epi-thelium and the extracellular matrix of the connective tissue isstained by alcian blue at both pH values (Fig. 1G, H).

Connective tissue fills the interior of the ridges and occupiesthe space between the epithelium and the outer muscularlayer. In general, the connective tissue of the crop in thisspecies is very poorly stained by aniline blue, the dye used inMasson’s trichrome to stain collagen. However, below theciliated epithelium of the large folds the connective tissue isstrongly stained by aniline blue (Fig. 1E). Close to the epi-thelium, the connective tissue is crossed by thin muscle fibres

(Fig. 1E), but in the outer layer of the crop wall muscle fibresare much thicker and transversely oriented (Fig. 1C).

Cells containing a very large vacuole, which was not stainedby any of the methods used, are abundant in the connectivetissue (Fig. 1D, E). Using series of semithin sections it waspossible to evaluate the three-dimensional form of these cells.In general, they have a round or oval shape, most of themwith an average diameter of 20–30 mm. The nucleus, locatedat the periphery of the cell, is usually flat and a very thin layerof cytoplasm surrounds the central vacuole, which occupiesabout 90% or more of the cell volume (Fig. 2A, B). Thesevacuolar cells are particularly abundant below the epithelium,occupying a substantial part of the connective tissue volume.Some are almost attached to the base of the epithelium

Figure 2. Semithin sections of the crop of Philinopsis depicta. A. Many vacuolar cells (asterisks) are present below the crop epithelium (ep), some ofthem in close contact with the base of the epithelium (arrows). The nucleus (arrowhead) is visible in one of the vacuolar cells, and severaltransversely sectioned muscle fibres (mf) can be seen in the connective tissue (ct). B. Vacuolar cells (asterisks) with a flat nucleus (arrowhead) arealso present among muscle fibres (mf) at the outer tissue layer of the crop wall. This figure appears in colour in the online version of Journal ofMolluscan Studies.

Figure 1. Anatomy, histology and histochemistry of the crop of Philinopsis depicta. A. General view of the digestive system showing the buccal bulb(bb), one of the salivary glands (arrowhead), the crop containing a shell of Bulla striata (cr), the posterior oesophagus (arrow) and the digestivegland (dg). B. The two large folds (arrows) are prominent structures in the posterior half of a dissected crop. C. Transverse section in the hind zoneof the crop stained with haematoxylin and eosin (HE), showing the ridges of the crop wall (arrows) and the two large folds (asterisks) forming achannel between them. The outer muscular layer is also visible (arrowheads). D. Detail of the crop wall showing the nonciliated epithelium(arrowhead) with secretory cells (arrows) and some vacuolar cells (asterisks) embedded in the connective tissue. HE staining. E. Masson’strichrome-stained section revealing the ciliated epithelium (arrowheads) lining the channel between the large folds. Many vacuolar cells (asterisks)can be seen in the connective tissue (ct) stained by aniline blue. Some muscle fibres are also visible (arrows). F. Secretory cells of the cropepithelium (arrows) are strongly stained by PAS reaction. G. At pH 2.5 the secretory cells (arrows) and the material covering the epithelium(arrowhead) are stained by alcian blue. H. Secretory cells (arrow) are weakly stained by alcian blue at pH 1, but the material covering theepithelium presents a stronger coloration (arrowhead). I. The secretory cells (arrow) are not stained by the tetrazonium coupling reaction.


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Figure 3. Ultrastructure of crop epithelial cells of Philinopsis depicta. A. Nonciliated epithelial cells are covered by short microvilli (arrowheads)embedded in a layer of extracellular material (arrow). Several lysosomes (ly) and mitochondria (mi) are visible in the cytoplasm above thenucleus (nu). B. Aggregates of glycogen granules stained by tannic acid and uranyl acetate method (asterisks). C. Detail of the basal region of theepithelium showing a hemidesmosome (arrow) attached to a bundle of filaments (asterisk) and the thin basal lamina (arrowhead). D. The epithelialcells lining the channel between the large ridges of the crop possess cilia (arrows) with long roots (arrowheads), mitochondria (mi) and severallysosomes (ly) in the apical region.


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(Fig. 2A), but were never observed crossing it to be in contactwith the lumen of the crop. Although in smaller numbers, thevacuolar cells are also present in the outer region of the cropwall among the transverse muscle fibres (Fig. 2B).

Histologically the posterior oesophagus is similar to the hindregion of the crop, with a continuation of the ciliated groovebetween two folds, which in this part of the digestive tube arenot significantly higher than the ridges lined by a nonciliatedepithelium. In the ciliated groove secretory cells are notpresent, but several cells secreting acid polysaccharides areinserted in the nonciliated epithelium, as in the crop. Vacuolarcells are also present in the connective tissue of the posterioroesophagus.

Ultrastructure and cytochemistry

The nonciliated crop and oesophageal epithelial cells possessshort apical microvilli embedded in a layer of low-densityextracellular material (Fig. 3A). The supranuclear region ofthese cells contains many vesicles, most of them withelectron-dense material, looking like lysosomes. Lipid dropletswere rare in these cells and the amount of glycogen granulesranged from abundant to scarce depending on the animals(Fig. 3B). Many and deep interdigitations are formed betweenneighbouring epithelial cells and in the basal region hemides-mosomes are frequent (Fig. 3C). The epithelial cells in theciliated channel are similar to the cells described above, butcontaining cilia with long ciliary roots, and more mitochondriaseem to be present in the apical region (Fig. 3D).

The secretory cells of the crop and posterior oesophagus arecharacterized by the presence of many large vesicles with lowelectron density (Fig. 4A). In matured cells, these vesicles fillmost of the dilated cytoplasm and fusion among them forms alarge mass of secretion. The basal region of these cells com-prises the irregularly shaped nucleus and several Golgi stacksformed by many flat cisternae with low electron-densitycontent (Fig. 4A). Using the tannic acid and uranyl acetatestaining method for polysaccharide detection, the secretory ves-icles became highly electron-dense (Fig. 4B). These cells werenever observed in the ciliated epithelium.

In both crop and posterior oesophagus, the extracellularmatrix of the connective tissue is very electron-lucent and con-tains a reduced amount of collagen fibrils (Fig. 4C), exceptbelow the ciliated epithelium of the large folds where thesefibrils are more abundant. The vacuolar cells found in the con-nective tissue possess a very large electron-lucent centralvacuole with some flocculent material, surrounded by an extre-mely thin cytoplasm (Fig. 4C). Tubular invaginations of thecell membrane are frequent in these cells (Fig. 5A), which alsocontain vesicles of different dimensions with flocculentmaterial. The vesicles can be open to the central vacuole(Fig. 5B). A thin basal lamina surrounds these cells (Fig. 5A).Rarely, cells with several smaller vacuoles were observed(Fig. 5C). In these cells, several mitochondria and tubularmembrane structures can be found in the cytoplasm. Thenucleus is peripheral and frequently with a kidney-shapedsection (Fig. 5C), as in the cells with a single large centralvacuole (Fig. 4C).

DISCUSSION

Anatomical and molecular data suggest that carnivory evolvedindependently more than once within cephalaspideans, but theexact number of evolutionary shifts is still uncertain(Mikkelsen, 1996; Malaquias et al., 2009). According toMalaquias et al. (2009) the radiation of cephalaspideans doesnot seem to be closely correlated with the reduction or loss ofthe shell, with lifestyle, with prey habitats or with digestive

anatomy. Instead, a correlation between phylogeny and aspecialization on vegetal or animal food sources was suggested(Malaquias et al., 2009). Furthermore, no obvious relationbetween food type and anatomical features was found(Malaquias et al., 2009). However, ultrastructural and histo-chemical studies might reveal particular traits of digestivesystem organs in different clades of carnivorous and herbivor-ous cephalaspideans that were undetected in previous anatom-ical studies. This article is the first to report histochemical andultrastructural aspects of the crop and oesophagus in carnivor-ous cephalaspideans, but many more species and other diges-tive system organs have to be investigated with these methodsbefore new attempts can be made to establish correlationsbetween digestive system features and molecular phylogeneticdata or food type.

In Philinopsis depicta, the two large folds at the posterior halfof the crop are a conspicuous feature of this organ. They con-tinue through the posterior oesophagus creating a ciliatedgroove from the stomach to the central region of the crop. Alldata indicate that extracellular digestion in the crop of gastro-pods depends on the arrival of a digestive fluid coming fromthe stomach containing enzymes secreted by the digestivegland, and that digestion products must flow out of the crop tobe absorbed mainly by cells of the digestive gland (Rudman,1972b; Dimitriadis, Hondros & Pirpasopoulou, 1992). Foldsforming a ciliated groove were also observed at the hind end ofthe crop in Philinopsis taronga, but in the aglajid Melanochlamyscylindrica similar structures were not reported (Rudman,1972b). According to Rudman (1972b) in P. taronga the cilia inthe groove beat towards the crop, suggesting that the fluidwith digestive gland enzymes is carried to the crop along thischannel. Thus, a ciliated channel extending from the stomachto the middle of the crop in P. depicta is probably important foran efficient release of digestive gland enzymes in the crop con-taining a large prey.

The cells observed in the crop and oesophageal epitheliumof the carnivore P. depicta are not substantially different fromthe ones described in the oesophagus and crop of the herbivor-ous opisthobranchs Bulla striata and Aplysia depilans(Lobo-da-Cunha & Batista-Pinto, 2005; Lobo-da-Cunha et al.,2010a, b). In gastropods, oesophageal and crop epithelial-supporting cells typically possess several lysosomes in the cyto-plasm above the nucleus, a border of microvilli and in somecases cilia (Bush, 1989; Boer & Kits, 1990; Leal-Zanchet,2002). In epithelial cells of the oesophagus of B. striata andcrop of A. depilans, arylsulphatase activity was detected inelectron-dense lysosomes (Lobo-da-Cunha & Batista-Pinto,2005; Lobo-da-Cunha et al., 2010a) which show the same mor-phology as the lysosome-like structures observed in epithelialcells of the crop of P. depicta. These lysosomes are naturallyinvolved in the intracellular digestion of substances collectedby endocytosis (Boer & Kits, 1990; Bourne et al., 1991). Inmolluscs the digestive gland is the major organ involved indigestion. In this organ, the digestive cells collect by endocyto-sis the nutritive particles arriving from the stomach, and thefinal stages of digestion occur in the lysosomes that are abun-dant and large in these cells (Walker, 1972; Lobo-da-Cunha,2000). However, intracellular digestion and nutrient absorp-tion have been reported in the oesophagus and crop of somegastropods (Walker, 1972; Bush, 1989; Payne & Crisp, 1989;Bourne et al., 1991) and also seem to occur in P. depicta.Another function of digestive tract epithelial cells is theaccumulation of reserves (Roldan, 1987; Boer & Kits, 1990;Dimitriadis et al., 1992; Lobo-da-Cunha & Batista-Pinto,2005). Glycogen is the main reserve substance in the epithelialcells in the crop of P. depicta, and the high variability in theamount of glycogen granules could be correlated with thenutritional status of each animal.


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Figure 4. Ultrastructure of crop secretory and vacuolar cells of Philinopsis depicta. A. Longitudinal section of a secretory cell showing the basalnucleus (nu), several Golgi stacks (Gs) and many electron-lucent vesicles (asterisks). Microvilli are present at the cell apex (arrow). B. Thesecretory vesicles are strongly stained by tannic acid and uranyl acetate method (asterisks). C. Vacuolar cells have a very thin cytoplasm (arrows)surrounding the large electron-lucent central vacuole (va) with some flocculent material. The connective tissue matrix is electron-lucent with fewcollagen fibrils (arrowheads). Abbreviations: nu, nucleus; mc, muscle cell.


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According to the histochemical data, the crop secretory cellsof P. depicta are of the mucous type that secretes acid polysac-charides, but their secretory vesicles do not possess detectableamounts of protein. Because alcian blue staining is stronger at

pH 2.5 than at pH 1, acid carboxylic groups must be moreabundant than sulphated groups in these polysaccharides(Jones & Reid, 1973). Thus, their secretion seems to have alubricating or protective role for the epithelium and not a

Figure 5. Ultrastructure of vacuolar cells of Philinopsis depicta. A. Vacuolar cells are surrounded by a thin basal lamina (arrowheads) and formmany tubular cell membrane invaginations (arrows). B. Some vacuolar cells possess thicker zones of cytoplasm containing vesicles (ve) that can beopened to the central vacuole (arrow). C. Cell containing several vacuoles (va), some mitochondria with an electron-dense matrix (mi) and tubularmembranar structures (arrows) in the cytoplasm. Abbreviation: nu, nucleus.


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digestive activity. These cells seem to correspond to the largespherules reported by Rudman (1972b) in the crop epitheliumof P. taronga. On the other hand, Rudman (1972b) did not findsecretory cells in the crop and stomach of the aglajidMelanochlamys cylindrica. In the herbivorous cephalaspideanBulla striata, the anterior oesophagus and first part of the pos-terior oesophagus also contain acid-polysaccharide-secretingcells and in the second part of the posterior oesophagus thesecretory cells produce a mixture of proteins and acid polysac-charides (Lobo-da-Cunha et al., 2010a, b). Mucous cells havealso been reported in the oesophagus of other carnivorous andherbivorous cephalaspideans (Fretter, 1939; Rudman, 1972a).Thus, according to the available data, in cephalaspideansthere is no evidence of a relationship between diet (carnivorousor herbivorous) and the secretory cells present in theoesophagus or crop.

The vacuolar cells are a remarkable feature of the connectivetissue of the crop of P. depicta. The high number of cell mem-brane invaginations in these cells suggests an intense endocyticactivity and the formation of vesicles transporting substancescollected from the surrounding connective tissue matrix to thelarge vacuole. The cells with several vacuoles have all the fea-tures of an earlier stage in the development of the vacuolarcells, before the fusion of the smaller vacuoles to create thelarge single vacuole. To the best of our knowledge, similar cellshave not previously been reported in the digestive tract ofother opisthobranchs; the presence of a large number of vacuo-lar cells could be a special trait of the crop of carnivorouscephalaspideans or just of aglajids.

The channel cells from the skin of the terrestrial slugAriolimax columbianus have a large central channel or reservoirfilled with fluid surrounded by a thin layer of cytoplasm(Luchtel, Martin & Deyrup-Olsen, 1984), as in the vacuolarcells of P. depicta. The cytoplasm of the channel cells containsmany tubular structures opened to the exterior and to theinterior reservoir (Luchtel et al., 1997), resembling the tubularcell membrane invaginations of the vacuolar cells of P. depicta.Ink injection experiments showed that the channel cells ofA. columbianus are capable of collecting ink particles from thesurrounding connective tissue and transfer them to the interiorreservoir via cytoplasmic tubules. However, the apex of thechannel cells crosses the epithelium, being able to release thecontent of the reservoir when stimulated to secrete (Luchtelet al., 1984, 1997). The vacuolar cells in the crop of P. depictaare abundant below the epithelium, but were never seen incontact with the lumen of the crop, and in this case release ofthe fluid contained in the central vacuole to the lumen seemsimprobable. Moreover, vacuolar cells are also present in theexternal layers of the crop wall, distant from the lumen.

The rhogocyte (pore cell) is a connective-tissue cell typespecific to molluscs. These cells show several pouch-like cellmembrane invaginations bridged by cytoplasmic bars formingvery distinctive slits at the cell surface (Haszprunar, 1996;Albrecht et al., 2001), which were not observed in the vacuolarcells of P. depicta. In general, rhogocytes contain a prominentnucleus, numerous mitochondria, well-developed rough endo-plasmic reticulum and Golgi apparatus, which are character-istics of cells with high synthesis activity (Sminia, 1972;Haszprunar, 1996; Albrecht et al., 2001). These are not thecharacteristics of the vacuolar cells found in P. depicta,at least at the developmental stages that were observed.Electron-lucent vacuoles or electron-dense membrane-boundgranules can be present in rhogocytes, but a single centralvacuole occupying 90% or more of the cell volume has notbeen reported in typical rhogocytes (Haszprunar, 1996).

On the other hand, it has been proposed that the calciumcells of the connective tissue of molluscs are a specific andmodified type of rhogocyte (Haszprunar, 1996). These cells

can reach a large diameter and contain big vacuoles loadedwith concretions with typical concentric layers of calcium car-bonate and organic matter (Sminia et al., 1977; Luchtel et al.,1997). Some calcium cells contain a single large calcareousspherule surrounded by a very thin layer of cytoplasm, with aperipheral nucleus (Richardot & Wautier, 1972). A very largevacuole surrounded by a thin layer of cytoplasm and a periph-eral nucleus are features of the vacuolar cells in the crop ofP. depicta, but spherules were not visible within the vacuole ofthese cells. However, the calcium salts of the spherules can bedissolved during tissue processing (Richardot & Wautier, 1972;Sminia et al., 1977) and this could have happened in the cropof P. depicta. Nevertheless, in the absence of typical concretionsit is not possible to say that these vacuolar cells are calciumcells. In the freshwater pulmonate Lymnaea stagnalis, these con-cretions are abundant in calcium cells of the connective tissuelayers around several organs, but the calcium concretions dis-solve in animals subjected to respiratory acidosis (Sminia et al.,1977). Under acidosis, the vacuoles of calcium cells sometimesjust contain moderately electron-dense material (Sminia et al.,1977), also found in the vacuolar cells of P. depicta. The exper-iments with L. stagnalis exposed to CO2-enriched water suggestthat calcium cells are a source of ions for pH buffering of thehaemolymph (Sminia et al., 1977); this is a possible functionfor the connective tissue vacuolar cells in the crop of P. depicta.To conclude, the vacuolar cells here reported might belong toa family of connective tissue cells that include the calcium cellsof pulmonate gastropods and rhogocytes but, with the avail-able data, it is not possible to be certain about the functions ofthe vacuolar cells and the reason for their abundance in thecrop of aglajids. Moreover, the presence or absence of thesecells in the digestive tract might constitute a significant differ-ence between carnivorous cephalaspideans like P. depicta, whichcontain many vacuolar cells, and herbivorous cephalaspideanslike Bulla striata in which such cells seem to be absent(Lobo-da-Cunha et al., 2010a, b).

ACKNOWLEDGEMENTS

The authors thank Mr Joao Carvalheiro and Miss JoanaCarvalheiro for the reproduction of photomicrographs. We alsowish to thank Prof. Gerhard Haszprunar and Prof. HeikeWagele for the helpful suggestions in the discussion about thevacuolar cells. Rita Coelho holds a grant from the Fundacaopara a Ciencia e a Tecnologia, Portugal (BDE 15577/2005)and this work was supported by ICBAS and CIMAR.

REFERENCES

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Microscopical study of the crop and oesophagus of the carnivorous opisthobranch Philinopsis depicta (Cephalaspidea, Aglajidae)

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