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Alcian blue staining for acidic polysaccharides, at pH 1.0 and 2.5, fol- lowed by nuclear ... The prostate was yellow, and a thin line separating the proximal and.
Received: 1 September 2017

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Revised: 27 November 2017

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Accepted: 7 December 2017

DOI: 10.1002/jmor.20790

RESEARCH ARTICLE

Histological, histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda, Cephalaspidea) Alexandre Lobo-da-Cunha1,2

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^ Angela Alves1 | Elsa Oliveira1 |

 nio E. Malaquias3 Manuel Anto 1 Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal 2

Interdisciplinary Center of Marine and Environmental Research (CIIMAR), 4450-208 Matosinhos, Portugal 3

Phylogenetic Systematics and Evolution Research Group, Department of Natural History, University Museum of Bergen, University of Bergen, Bergen, Norway Correspondence A. Lobo-da-Cunha. Department of Microscopy, Institute of Biomedical Sciences Abel Salazar. Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Email: [email protected]

Abstract Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide-secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be

Funding information Institute of Biomedical Sciences Abel Salazar (ICBAS) of the University of Porto

involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non-European species attributed to this genus.

KEYWORDS

microscopy, Mollusca, opisthobranchs, reproductive system, ultrastructure

1 | INTRODUCTION

with or without external shell, occurring worldwide from polar to tropical seas, in shallow and deep waters (Malaquias, Bericibar, & Reid,

Due to the biological and systematic relevance of the reproductive sys-

2009; Malaquias, Dodds, Bouchet, & Reid, 2009; Oskars, Bouchet, &

tem, the general anatomy of reproductive organs is often reported in

Malaquias, 2015). Cephalaspideans are hermaphroditic and their repro-

taxonomic descriptions of gastropods. However, a deeper knowledge

ductive system presents some peculiar aspects. Sperm cells formed in

about these organs requires detailed histological studies and even

the gonad (ovotestis), which is associated with the digestive gland, flow

ultrastructural observations that so far have been done only for a few

through the ampulla, ampullar duct, and common genital duct to reach

species of Cephalaspidea gastropods (Amorim, Oliveira, Malaquias, &

the common genital aperture on the right side at the middle of the

Lobo-da-Cunha, 2013; Klussmann-Kolb, 2001). With more than 600

body. From there, spermatozoa travel through the ciliated external

species, the clade Cephalaspidea is the species-rich among the euopis-

seminal groove entering into the male copulatory apparatus, located on

thobranch gastropods, including carnivorous and herbivorous species

the right side of the head. This apparatus includes the copulatory organ

Journal of Morphology. 2018;1–12.

wileyonlinelibrary.com/journal/jmor

C 2018 Wiley Periodicals, Inc. V

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(penis or penial papilla) and the prostate where the spermatozoa stay until copulation (Amorim et al., 2013; Malaquias & Reid, 2008).

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2.2 | Tissue processing for light and electron microscopy

The genus Haminoea, Turton and Kingston, 1830, belongs to the Haminoeidae, the most diverse of the Cephalaspidea, and comprises herbivorous species with a thin external bubble-shaped shell that is more or less covered by the parapodial lobes of the animal. The species of that genus are distributed throughout temperate and tropical regions of the world (Rudman, 1971). However, in a recent molecular phylogenetic study of cephalaspideans, this genus as traditionally defined was not recognized as monophyletic and it was questioned whether the traditional Haminoea from the Atlantic and Indo-Pacific belong to the same genus (Oskars et al., 2015). Haminoea navicula (da Costa, 1778) is one of the seven species recognized as native to Europe. It is found in the Mediterranean Sea and in the Atlantic from southern England and Ireland to the Strait of Gibraltar, living on softsediment bottoms from intertidal areas down to a depth of 4 m in coastal lagoons and estuaries (Malaquias & Cervera, 2006). In addition to the native species, H. japonica, Pilsbry, 1895 (5 H. callidegenita) and H. cyanomarginata, Heller and Thompson, 1983 from the Indo-Pacific oceans, were reported on European coasts (Crocetta & Vazzana, 2009; Hanson et al., 2013). The taxonomy of Haminoea is notoriously difficult because many

For histological sections, penial sheath, seminal duct, and prostate collected from two animals were fixed for 24 hr with Davidson solution made with seawater, dehydrated in a graded series of ethanol and embedded in paraffin. Sections from both animals, with a thickness of 6 mm, were stained with hematoxylin and eosin, and with Masson’s trichrome. For semithin and ultrathin sections, samples collected from 3 animals were fixed for about 2 hr at 48C in 2.5% glutaraldehyde and 4% formaldehyde (obtained from hydrolysis of para-formaldehyde), diluted with 0.4 mol l21 cacodylate buffer pH 7.4 (final buffer concentration 0.28 mol l21). After washing in buffer, they were postfixed with 2% osmium tetroxide buffered with cacodylate, dehydrated in increasing concentrations of ethanol and embedded in Epon. Semithin sections obtained from samples of the three animals, with a thickness of 2 lm, were stained with methylene blue and azure II for light microscopy. Ultrathin sections with a thickness about 80 nm were stained with uranyl acetate and lead citrate, before being observed in a JEOL 100CXII transmission electron microscope.

2.3 | Histochemistry

species were described only from shells, which are in most cases simi-

Alcian blue staining for acidic polysaccharides, at pH 1.0 and 2.5, fol-

lar between species, and with few exceptions, their color patterns are

lowed by nuclear staining with hematoxylin was applied to deparaf-

also not much variable (Garabedian et al., 2017; Malaquias & Cervera,

finized sections of penial sheath, seminal duct, and prostate

2006; Schaefer, 1992; Thompson, 1981). Consequently, modern taxo-

obtained from both animals whose male copulatory apparatus were

nomic work on the genus relies in detailed internal anatomy and

fixed with Davidson solution. The tetrazonium coupling reaction for

molecular data, and the male copulatory organs are considered of high

protein detection, the PAS reaction for polysaccharides, and the

taxonomic value (Chaban & Chernyshev, 2016; Gibson & Chia, 1989;

Schmorl’s method for melanin and other reducing substances (Gan-

Malaquias & Cervera, 2006; Rudman, 1971; Talavera, Murillo, & Tem-

ter & Jollès, 1970) were applied to 2 lm thick semithin sections of

plado, 1987). The morphology of reproductive systems is in general of

Epon embedded samples obtained from three animals. For the tetra-

great importance in the systematics and phylogenetic relationships of

zonium coupling reaction and the Schmorl’s method, the embedding

gastropod molluscs (e.g. Ghiselin, 1965; Klussmann-Kolb, 2001; Ponder

medium was removed from the semithin sections using an alcoholic

& Lindberg, 1997) and therefore knowledge of their morphology,

solution of sodium ethoxide, prepared dissolving sodium hydroxide

homology and function is important, but often these systems are not

to saturation in absolute ethanol (Lane & Europa, 1965). Subse-

fully documented, and the pathways of spermatozoa and ova are far

quently, sections were thoroughly washed in absolute ethanol and

from understood (Amorim et al., 2013; Malaquias & Reid, 2008).

in water. For the tetrazonium reaction, the semithin section were

Therefore, to extend the current knowledge about the reproductive

oxidized with 0.06% hydrogen peroxide for 10 min to remove

system of haminoid cephalaspideans, the male copulatory apparatus of

osmium tetroxide, and washed with water before treatment with a

H. navicula was investigated with light and transmission electron

freshly-prepared 0.2% solution of fast blue salt B in veronal-acetate

microscopy (TEM).

buffer (pH 9.2) for 10 min. After washing, sections were treated for 15 min with a saturated solution of b-naphthol in veronal-acetate

2 | MATERIALS AND METHODS 2.1 | Animals and collection site

buffer (pH 9.2) and washed. For melanin detection, sections were treated for 10 min with a freshly prepared Schmorl’s solution containing ferric chloride and potassium ferricyanide, and afterward washed with water. For PAS reaction, sections were oxidized with

Five specimens of Haminoea navicula (da Costa, 1778) were collected

1% periodic acid for 10 min, washed with water, stained with Schiff

in May, during low tide in Ria de Aveiro, a coastal lagoon system con-

reagent for 20 min and washed again. Semithin sections without

nected to the Atlantic Ocean on the central Portuguese shoreline. After

Epon were dehydrated through a graded series of ethanol solutions,

removal of the male copulatory apparatus, the penial sheath, seminal

cleared with xylene and mounted with Coverquick 2000 mounting

duct, and prostate were separated and processed for light and electron

medium. Semithin sections with Epon (PAS reaction) were air dried

microscopy as reported below.

before being mounted.

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3 | RESULTS

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proximal and distal lobes (Figure 2c) and continues through the seminal duct. Openings in this epithelium allow the secretion coming from the

3.1 | General morphology, histology, and histochemistry

tubules to flow into the lumen of the prostate (Figure 2c inset). The tubules of the proximal lobe could be divided in three zones (Figure 2d). The basal zone contained a smaller cell type that secreted acid

The male copulatory apparatus of H. navicula comprises the penis within a penial sheath, seminal duct, and a bilobed prostate (Figure 1a). The tip of the penis covered with small spikes could be seen protruding from the penial sheath, revealing a subapical slit where the penial duct opens to the exterior (Figure 1b). Additionally, black pigment spots were externally visible on the sheath that covers the penis (Figure 1b). The prostate was yellow, and a thin line separating the proximal and distal lobes of this organ was evident by inspection with the stereo microscope (Figure 1a). The penis is a muscular structure with a tip covered by numerous spikes consisting of densely packed muscular cells externally coated by a thin cuticle (Figure 1c,d). The duct that runs through the penis was lined by a ciliated epithelium under which vacuolar cells were observed. The number of vacuolar cells increased toward the distal portion of the penis, where a large number of densely packed vacuolar cells could be observed beneath the ciliated epithelium of the penial duct. Some of these cells were more deeply embedded in the surrounding muscular tissue (Figure 1e). The penial sheath consists of muscular tissue folds (Figure 1c), internally lined by an epithelium formed by short columnar cells with an apical border of microvilli (Figure 1f). Below this epithelium, several polysaccharide-secreting cells and pigment cells were observed. In semithin sections stained with methylene blue and azure II the polysaccharide-secreting cells showed metachromatic vesicles (Figure 1f), which were strongly PAS positive (Figure 1g) but not stained by Alcian blue. Although a few polysaccharide-secreting cells could be seen in the epithelium, the vast majority of them were below the epithelium having a thin neck that crossed the epithelium to release the secretion on the surface (Figure 1g). The pigment cells, which were darker than the surrounding tissues, had irregular cytoplasmic extensions projecting from the main cell body (Figure 1h) and in semithin sections were stained by the Schmorl’s method for melanin detection (Figure 1i). The seminal duct connecting the penis with the prostate was

polysaccharides and a larger cell type that secreted proteins. The first ones contained small secretory vesicles that were strongly metachromatic in semithin sections stained with methylene blur and azure II (Figure 2e). These cells were stained by Alcian blue at pH 1.0 and 2.5 (Figure 2f) and by the PAS reaction (Figure 2g), but proteins were not detected in their secretory vesicles (Figure 2h). Cells of the second type contained large secretory vesicles that stained light blue with methylene blue and azure II (Figure 2e), being neither stained by Alcian blue or PAS reaction (Figure 2f,g). On the other hand, proteins were detected by the tetrazonium reaction in the secretory vesicles of these cells (Figure 2h). The middle zone of the proximal prostate tubules was characterized by secretory cells with large vacuoles in the apical region (Figure 3a) containing scattered bits of glycoproteic material that were stained both by PAS (Figure 3b) and tetrazonium reactions (Figure 3c), with a stain intensity ranging from moderate to strong. A few polysaccharide secreting cells like the ones found at the basal zone also appeared in the middle zone, but were much less stained (Figure 3a). The apical zone of these tubules was the longest, being mainly formed by tall columnar cells containing a basal nucleus and several large secretory vesicles that stained light blue in semithin sections stained with methylene blue and azure II (Figure 3d). These secretory vesicles were weakly stained by the PAS reaction (Figure 3e), but moderately stained by the tetrazonium reaction for protein detection (Figure 3f). Small ciliated cells with unstained vesicles and an elongated nucleus could be seen between the large secretory cells in the apical zone of proximal prostate tubules (Figure 3d). The tubules of the distal lobe of the prostate contained secretory cells of just one type, whose secretory vesicles stained dark blue with methylene blue and azure II (Figure 3g) and were very strongly stained by both PAS (Figure 3h) and tetrazonium reactions (Figure 3i). In the distal lobe of the prostate, no cells were stained by Alcian blue (Figure 2f). Small ciliated cells were also observed in the tubules of the distal lobe of the prostate. The staining properties of the prostate secretory cells are summarized in Table 1.

encircled by a thick muscular wall. In this part of the male copulatory apparatus, vacuolar cells were also abundant beneath the ciliated epithelium of the seminal duct and some of these cells were deeply

3.2 | Ultrastructure

embedded in the muscular wall (Figure 1j). In the vacuolar cells, the

Transmission electron microscopy was conducted to characterize the

nucleus was pushed to the margin by the large vacuole that filled most

ultrastructure of the cell types previously studied by light microscopy

of the cell volume (Figure 1k). Material inside this vacuole was stained

in the male copulatory apparatus of H. navicula. In the penial sheath,

by Alcian blue at both pH 2.5 and 1.0, revealing a strongly acidic pH

polysaccharide-secreting cells were frequently found between the epi-

(Figure 1l).

thelium and the muscular tissue. Nevertheless, the ultrastructural

The two lobes of the prostate were separated by a thin muscular

observations confirmed that these secretory cells could also occur in

septum with a central opening through which the lumen of the two

the epithelium of the penial sheath (Figure 4a). Moreover, they had the

lobes were connected. The proximal lobe is the smallest, corresponding

same general ultrastructural features regardless of being part of the

to about one third of the organ, and both lobes consisted of numerous

epithelium or being located below it. In the apical region, these cells

tubules formed by secretory cells that discharge their products into the

contained many secretory vesicles with low electron-density. Some

prostate lumen (Figure 2a,b). The lumen of the proximal lobe is lined by

Golgi stacks and rough endoplasmic reticulum cisternae were found

a ciliated epithelium, which starts at the connection point between the

around the nucleus and in the basal region of these secretory cells

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F I G U R E 1 Haminoea navicula, stereo microscope images (a, b), histological sections stained with hematoxylin and eosin (c, j), Masson’s trichrome (d, e) and alcian blue (l), and semithin sections stained with methylene blue and azure II (f, h, k), PAS reaction (g), and Schmorl’s method (i). (a) Male copulatory apparatus. (b) The protruded penis reveals a subapical slit (arrowhead), and pigment cells (arrow) are visible on the penial sheath. (c) The penial sheath covers the penis with spikes on the tip (arrow). The external opening of the penial duct is also visible (arrowhead). (d) The penial spikes coated by a thin cuticle (arrows) consist of densely packed muscular cells whose nuclei are visible (arrowheads). (e) The ciliated epithelium of the penial duct (arrowheads) is surrounded by vacuolar cells (arrows). (f) Secretory cells containing metachromatic vesicles (arrows) below the epithelium with a border of microvilli (arrowheads) in the penial sheath. (g) Secretory cells of the penial sheath stained by PAS reaction (arrows), revealing the thin neck (arrowheads) that crosses the epithelium. (h) A pigment cell (arrows) below the epithelium of the penial sheath. (i) Pigment cells (arrows) stained by the Schmorl’s method for melanin detection. (j) Transverse section of the seminal duct showing many vacuolar cells (arrows) surrounding the epithelium. (k) Vacuolar cells (asterisks) with a marginal nucleus (arrowheads) below the epithelium of the seminal duct. (l) Alcian blue staining in the vacuolar cells (arrows) of the seminal duct. dl, distal lobe of the prostate; ep, epithelium; mu, muscular cells; nu, nucleus; pe, penis; pl, proximal lobe of the prostate; ps, penial sheath; sd, seminal duct

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Haminoea navicula, light microscopy micrograph sections of the bilobed prostate stained with hematoxylin and eosin (a, c), Masson’s trichrome (b) and Alcian blue (f), and semithin sections stained with methylene blue and azure II (d, e), PAS reaction (g), and tetrazonium reaction (h). (a) Longitudinal section of the prostate showing the septum (arrowheads) separating the two lobes and the external muscular layer (arrows). (b) Muscular septum (arrows) separating the two lobes of the prostate. (c) A ciliated epithelium (arrowheads) lines the lumen of the prostate proximal lobe and the secretion flows through openings in this epithelium (arrow, inset). (d) The tubules that form the proximal lobe of the prostate can be divided in basal (ba), middle (mi), and apical (ap) zones. (e) Basal region of the prostate proximal lobe tubules contains cells with metachromatic secretory vesicles (arrows) and cells with light blue secretory vesicles (arrowheads). (f) Alcian blue staining revealing acid polysaccharide-secreting cells (arrows) in the basal region of the prostate proximal lobe tubules. (g) PAS reaction stained polysaccharide secreting cells (arrows) of the basal region of the prostate proximal lobe tubules, but the other cell type (asterisks) is not stained. (h) The tetrazonium reaction stains the vesicles of protein-secreting cells (arrowheads) in the basal region of the prostate proximal lobe tubules, but not the vesicles of polysaccharide-secreting cells (arrows). dl, distal lobe of the prostate; mu, muscular septum; nu, nucleus; pl, proximal lobe of the prostate

FIGURE 2

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Haminoea navicula, semithin sections of the prostate stained with methylene blue and azure II (a, d, g), PAS reaction (b, e, h), and tetrazonium reaction (c, f, i). (a) Cells with vacuoles containing secretory products (arrowheads) and a few cells with weakly stained metachromatic vesicles (arrow) in the middle zone of a prostate proximal lobe tubule. (b) Polysaccharide detection in secretion accumulated in the vacuoles (arrowheads) of cells in the middle zone of the proximal lobe tubules. (c) Protein detection in the secretion accumulated within the vacuoles (arrowheads) of cells in the middle zone of the proximal lobe tubules. Cell secreting polysaccharides are not stained (arrow). (d) Secretory cells (asterisks) of the apical zone of the proximal lobe tubules. Smaller cells with clear vesicles (arrowheads) fill the space between the secretory cells. (e) Polysaccharide detection in the vesicles (arrowheads) of the secretory cells that constitute the apical zone of the proximal lobe tubules. (f) Protein detection in the secretory vesicles (arrowheads) in the apical zone of the proximal lobe tubules. (g) Cells of the distal lobe of the prostate with apical secretory vesicles (arrowheads). (h) Polysaccharide detection in vesicles (arrowheads) of the prostate distal lobe secretory cells. (i) Protein detection in vesicles (arrowheads) of the prostate distal lobe secretory cells. nu, nucleus

FIGURE 3

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T AB LE 1

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Staining properties of the secretory products in prostate cells of H. navicula Methylene blue 1 azure II

Alcian blue pH 1.0 and 2.5 (acid polysaccharides)

PAS reaction (polysaccharides)

Tetrazonium reaction (proteins)

Polysaccharide secreting cells of the basal zone

Purple (metachromatic)

111

111

2

Protein secreting cells of the basal zone

Light blue

2

2

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Glycoprotein secreting cells of the middle zone

Dark blue

2

11

111

Glycoprotein secreting cells of the apical zone

Light blue

2

1

11

Dark blue

2

111

111

Cell types Proximal lobe tubules

Distal lobe tubules Glycoprotein secreting cells of the posterior lobe

Stain intensity: 2 unstained; 1 weak; 1 1 moderate; 1 1 1 strong.

(Figure 4a,b). Pigment cells of the penial sheath had the cytoplasm

similar to the protein secreting cells of the basal zone, although the

packed with vesicles and few organelles were visible. Some of these

dilated rough endoplasmic reticulum cisternae did not reach so large

vesicles were oval in shape and filled with electron-dense material, but

dimensions (Figure 6b). Along the tubules of the proximal prostate

larger vesicles with scattered electron-dense material were much more

lobe, small ciliate cells were located between the large secretory cells

common (Figure 4c,d). The seminal duct epithelium was formed by cili-

and their cilia filled the lumen.

ated cells that contained numerous deep cell membrane infolds in the

In the prostate distal lobe, the cytoplasm of the secretory cells was

basal region, and many vacuolar cell were seen very close to basal lam-

mostly filled with dilated rough endoplasmic cisternae and some Golgi

ina of this epithelium (Figure 4e). In vacuolar cells, the nucleus always

stacks were also present. The rough endoplasmic cisternae could be

had a peripheral position, and an extremely thin cytoplasmic layer sur-

highly dilated and the secretory vesicles located above the round

rounded the huge vacuole of these cells (Figure 4f). A few mitochondria

nucleus were electron dense (Figure 6c). The tubules of the prostate

and rough endoplasmic reticulum cisternae were present, mostly in the

distal lobe also included ciliated cells, inserted between the secretory

vicinity of the nucleus of vacuolar cells where the cytoplasmic layer

ones. The nucleus and most of the cytoplasm of the ciliated cells were

was thicker (Figure 4g).

at the top of the epithelium, just with an extremely thin cytoplasmic

In the basal zone of the prostate proximal lobe tubules, the two

stalk reaching the base of the epithelium. Both cytoplasm and nucleus

types of secretory cells previously seen in semithin sections were also

were more electron-dense in the ciliated cells that in the secretory

observed by transmission electron microscopy. At the ultrastructural level, polysaccharide secreting cells were characterized by secretory

cells. Additionally, the ciliated cells contained several electron-lucent vacuoles that filled a large part of the cell volume (Figure 6d).

vesicles with low electron-density. Several Golgi stacks with thin cisternae were observed near the nucleus of these cells (Figure 5a). The protein secreting cells had many secretory vesicles with a fine granular

4 | DISCUSSION

content of high electron density, and their cytoplasm was filled with dilated rough endoplasmic reticulum cisternae containing granular material (Figure 5b). In some of these cells, dilated rough endoplasmic

The male copulatory apparatus was considered an important systematic feature to differentiate species within the genus Haminoea (Gibson

reticulum cisternae were huge, reaching areas of about 60–70 mm in

& Chia, 1989; Malaquias & Cervera, 2006; Rudman, 1971; Schaefer,

ultrathin sections. The content of the most dilated cisternae was less

1992; Talavera et al., 1987). However, despite the general morphology

electron dense than the content of the secretory vesicles, but had

of the male copulatory apparatus being documented in several studies

higher density than the less dilated rough endoplasmic reticulum cister-

about species of this genus, data on the internal anatomy and histology

nae of these cells (Figure 5c,d). Golgi stacks formed by thin cisternae

of its various structures have rarely been reported.

2

with a moderately electron-dense content and some small mitochon-

The penis presents some morphological differences among the

dria were also present. The secretory cells of the middle zone of the

Haminoea species native to Europe. In some of these species, the penis

proximal prostate lobe tubules were easily recognized by their large

have a smooth tip and in others an apical crest, but H. navicula is the

vesicles and vacuoles containing scattered portions of secretion. In

only species with numerous spikes on the tip of the penis (Alvarez, Gar-

some of these cells, the portions of secretory material were uniformly

cía & Villani, 1993; Malaquias & Cervera, 2006). In H. elegans, a species

electron-dense, in others an electro-dense external layer surrounded a

found in warm waters on both sides of the Atlantic, the penis has

less dense core with a finely granular structure (Figure 6a). The rough

numerous transverse folds along its length and a wide longitudinal

endoplasmic reticulum was very abundant and several Golgi stacks

sperm groove (Martínez & Ortea, 1997). However, a penial bulb lacking

formed by thin cisternae with electron-dense content were also pres-

a distinct penial papilla was reported in H. japonica (5 H. callidegenita).

ent in the cytoplasm of these cells. In the apical zone of the tubules of

Thus, even without detailed histological data about the male copulatory

the prostate proximal lobe, the secretory cells were ultrastructurally

apparatus of H. elegans and H. japonica, the penis of these species

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Haminoea navicula, transmission electron micrographs of the penial sheath and seminal duct cells. (a) Cells with an apical border of microvilli (arrowheads) and a polysaccharide-secreting cell (asterisk) form the epithelium of the penial sheath. (b) Secretory vesicles (asterisks), rough endoplasmic reticulum cisternae (arrowheads) and a Golgi stack (arrow) in a polysaccharide-secreting cell located below the epithelium of the penial sheath. (c–d) Pigment cell of the penial sheath containing electron-dense vesicles (arrows) and vesicles with scattered electron-dense material (asterisks). (e) Cell membrane infolds (arrows) in the basal region of the epithelium of the seminal duct. (f–g) Vacuolar cells of the seminal duct. Some mitochondria (arrows) can be seen in the thicker portion of the cytoplasm adjacent to the nucleus. ep, epithelium; mu, muscle cells; nu, nucleus; va, vacuolar cells

FIGURE 4

seems substantially different from the penis of native European spe-

described as a tubular sac containing an incurrent sperm groove. In H.

cies. In a study about the genus Haminoea, Rudman (1971) describes

zelandiae, this groove lined by a ciliated epithelium contains a complex

the male copulatory apparatus of three Indo-Pacific species (H. crocata,

of subepithelial gland cells, whereas the rest of the penis bulb is inter-

H. cymbalum, and H. zelandiae) and one species from the Atlantic coast

nally lined by an unciliated epithelium (Rudman, 1971). The internal

of North America (H. solitaria). These species have a penis bulb

structure of these penis bulbs seems very different from the compact

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Haminoea navicula, transmission electron micrographs of the two types of secretory cells in the basal zone of the prostate proximal lobe tubules. (a) Polysaccharide secreting cells contain secretory vesicles with low electron-density (asterisks). (b) Protein secreting cell with dilated rough endoplasmic reticulum cisternae and secretory vesicles (asterisks). (c) Protein secreting cell containing very large cisternae (asterisks) and less dilated cisternae of rough endoplasmic reticulum. (d) Detail of a very large rough endoplasmic reticulum cisternae (asterisk) showing the ribosomes (arrowheads). Gs, Golgi stacks; nu, nucleus; rer, rough endoplasmic reticulum

FIGURE 5

muscular penis of H. navicula. Although without a histological study,

The abundance of vacuolar cells below the epithelium of the semi-

the penis of H. orbignyana seems also to be a compact muscular struc-

nal duct suggests that these peculiar cells have an important physiolog-

ture (Malaquias & Cervera, 2006). Moreover, the absence of secretory

ical role in the male copulatory apparatus of H. navicula. Cells of the

cells in the seminal duct in H. navicula contrasts with the abundance of

same type were reported in the crop of the cephalaspideans Aglaja tri-

secretory cells reported in the incurrent sperm groove of H. zelandiae

colorata and Philinopsis depicta (Aglajidae). In these two species, vacuo-

(Rudman, 1971). However, secretory cells were abundant in the vesti-

lar cells were found in large numbers below the crop epithelium with

bule of the male copulatory apparatus of the cephalaspid Bulla striata

some of them more deeply embedded in the muscular tissue of the

(Bullidae), which contained three different types of secretory cells

crop wall (Lobo-da-Cunha et al., 2011, 2014), exactly like in the seminal

(Amorim et al., 2013). In H. navicula, small secretory cells of a single

duct of H. navicula. In the crop of A. tricolorata, calcium was detected in

type were found in the penial sheath, which is lined by an unciliated

the large vacuole of these cells (Lobo-da-Cunha et al., 2014), and

epithelium, but most secretory activity takes place in the prostate. The

experiments made with the freshwater pulmonate snail Lymnaea stag-

secretory activity inside the penial sheath of H. navicula could be

nalis suggest that cells containing calcium in vacuoles could be involved

involved in lubrication of this region.

in pH buffering (Sminia et al., 1977). Thus, in H. navicula, the vacuolar

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Haminoea navicula, transmission electron micrographs of the prostate. (a) Secretory cell from the middle region of the proximal prostate lobe tubules. A portion of secretory material (asterisk) is located inside a large vacuole also containing flocculent material. (b) Secretory cells of the apical zone of the prostate proximal lobe tubules contain many dilated rough endoplasmic reticulum cisternae, some Golgi stacks, and secretory vesicles (asterisks). (c) Dilated rough endoplasmic reticulum cisternae and large highly electron-dense vesicles (asterisks) in a secretory cell of the prostate distal lobe. Golgi stacks are also visible (arrows). (d) Ciliated cells with electron-lucent vacuoles fit between the secretory cells (asterisks) in the tubules of the prostate distal lobe. Their cilia fill the lumen of the tubule. ci, cilia; Gs, Golgi stack; nu, nucleus; rer, rough endoplasmic reticulum; va, vacuoles

FIGURE 6

cells may store ions in the vacuole to be used for ion and pH regulation

in H. navicula, H. exigua, H. orbignyana, H. orteai, and H. templadoi, but

in the seminal duct. Moreover, the abundance of basal cell membrane

separated by a narrow region in H. hydatis and H. fusari (García, Perez-

invaginations in the epithelium of the seminal duct is consistent with

mez, 1991; Malaquias & Cervera, 2006). The Hurtado, & García-Go

an intense transport of ions between this epithelium and the vacuolar

prostate also has two lobes in H. vesicula from the Pacific coast of

cells just below. Vacuolar cells were not found in the male copulatory

North America (Gibson & Chia, 1989) and in H. elegans from the tropi-

apparatus of B. striata (Amorim et al., 2013), emphasizing the histologi-

cal Atlantic (Martínez & Ortea, 1997). The prostate is not divided into

cal and physiological differences between the male copulatory appara-

lobes in other Indo-Pacific species (Gibson & Chia, 1989; Gosliner &

tus in different families of cephalaspideans.

Behrens, 2006; Rudman, 1971), although in H. cymbalum an accessory

As in the other six native European species of the genus Haminoea,

gland attached to the prostate may correspond to a proximal lobe (Rud-

the prostate of H. navicula has two lobes. These lobes are contiguous

man, 1971). However, histological and histochemical data are not

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available for other species of this genus to allow a comparison with the

ORC ID

cells of H. navicula prostate. The light microscopy staining techniques

Alexandre Lobo-da-Cunha

11

http://orcid.org/0000-0003-4606-9209

applied to paraffin and semithin sections were valuable for the characterization of the prostate secretory cells in H. navicula. The histological and histochemical study revealed the complexity of this organ with five types of secretory cells, which seem to secrete different substances that probably are involved in the maintenance and eventually also in the maturation of spermatozoa. The proximal lobe is more complex than the distal lobe, and each lobe seems to produce a different type of glycoproteic secretion. The prostate of B. striata was found to be very different, consisting on a single coiled tube containing secretory cells of just one type (Amorim et al., 2013). Spermatozoa were found inside the prostate of B. striata (Amorim et al., 2013), but not in the prostate of H. navicula specimens used for this study. It can be concluded that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non-European species attributed to this genus (Gosliner & Behrens, 2006; Martínez & Ortea, 1997; Rudman, 1971). In a phylogenetic tree of cephalaspideans based on two mitochondrial genes (COI and 16S rRNA) and two nuclear genes (28S rRNA and histone3), the Indo-Pacific species H. cymbalum and H. ovalis stood closer to an unidentified Smaragdinella species from Singapore than to the European species H. orbignyana and H. hydatis (Oskars et al., 2015). These data hint at a possible different genetic affiliation of IndoPacific species, but this remains to be proved. Unfortunately, an available phylogenetic hypothesis including a larger number of worldwide Haminoea species, based on one mitochondrial gene and one nuclear gene (COI and 28S rRNA), does not allow to test of the monophyly of the genus Haminoea because other genera of Haminoeidae were not included (Hanson et al., 2013). Thus, a more comprehensive phylogenetic study of the family Haminoeidae and Haminoea genus (sensu Burn & Thompson, 1998) including better gene coverage and morphological characters will be necessary to clarify the monophyly and evolution of morphological traits in these snails. Due to the relevance of copulatory organs in reproductive biology and isolation, histological studies of male copulatory apparatus in a larger number of species will certainly contribute to improve understanding of the systematics, homology, and evolutionary adaptations in the family Haminoeidae.

AC KNOW LE DGME NT S This research was funded by the Institute of Biomedical Sciences Abel Salazar (ICBAS) of the University of Porto. The authors thank Prof. Gonçalo Calado (Department of Life Sciences, Lusophone University of Humanities and Technologies, Lisbon, Portugal) for providing the specimens used in this study.

AUT HOR C ONTR IB UTIONS ALC and MAEM conceived and designed the study. ALC performed laboratorial work and wrote the manuscript with inputs from MAEM. EO and AA performed laboratorial work.

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^ Oliveira E, How to cite this article: Lobo-da-Cunha A, Alves A, Malaquias MAE. Histological, histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda, Cephalaspidea). Journal of Morphology. 2018;00:1–12. https://doi.org/10.1002/jmor.20790