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Fine structure of Mytella falcata (Bivalvia) gill filaments

Jose Augusto de Oliveira David a,*, Renato B. Salaroli b, Carmem S. Fontanetti a

a Departamento de Biologia, Instituto de Biociencias, Universidade Estadual Paulista, Av. 24A, no. 1515, CP 199,

CEP 13506-900, Rio Claro, SP, Brazilb NAP/MEPA, ESALQ, Universidade de Sao Paulo, Av. Padua Dias, no. 11, CP 9, CEP 13418-900, Piracicaba, SP, Brazil

Received 4 April 2007; received in revised form 9 June 2007; accepted 10 June 2007

Abstract

Bivalve filter feeders are sessile animals that live in constant contact with water and its pollutants. Their gill is an organ highly exposed to these

conditions due to its large surface and its involvement in gas exchanges and feeding. The bivalve Mytella falcata is found in estuaries of Latin

America, on the Atlantic as well as the Pacific Coast. It is commonly consumed, and sometimes is the only source of protein of low-income

communities. In this study, gill filaments of M. falcata were characterized using histology, histochemistry and transmission electron microscopy for

future comparative studies among animals exposed to environmental pollutants. Gill filaments may be divided into abfrontal, intermediate and

frontal zones. Filaments are interconnected by ciliary discs. In the center of filaments, haemocytes circulate through a haemolymph vessel

internally lined by an endothelium and supported by an acellular connective tissue rich in polysaccharides and collagen. The abfrontal zone

contains cuboidal cells, while the intermediate zone consists of a simple squamous epithelium. The frontal zone is composed of five columnar cell

types: one absorptive, mainly characterized by the presence of pinocytic vesicles in the apical region of the cell; one secretory, rarely observed; and

three ciliated with abundant mitochondria. All cells lining the filament exhibit numerous microvilli and seem to absorb substances from the

environment. PAS staining was observed in mucous cells in the frontal and abfrontal zones. Bromophenol blue allowed the distinction of

haemocytes and detection of a glycoprotein secretion in the secretory cells of the frontal region. The characteristics of M. falcata gill filaments

observed in this study were very similar to those of other bivalves, especially other Mytilidae, and are suitable for histopathological studies on the

effect of water-soluble pollutants.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Histochemistry; Polysaccharides; Protein; Collagen; Transmission electron microscopy; Gill morphology; Mussel

1. Introduction

The bivalve Mytella falcata is found on the Atlantic Coast,

from Venezuela to Argentina, as well as on Pacific shores and

the Galapagos Islands (Narchi and Galvao-Bueno, 1983).

Considered an important food item, this species is widely

consumed in several northeastern regions of Brazil and is

sometimes the only source of protein of lower-income

communities (Boffi, 1979). Species of the genus Mytella are

of great interest in biomonitoring studies, since they are found

partially buried in estuaries and thus provide data on the

conditions of the substrate (Narchi and Galvao-Bueno, 1983).

In bivalve mollusks such as the mussel Mytilus gallopro-

vincialis, the gills are key organs involved in nutrient uptake,

digestion and respiration (Gomez-Mendikute et al., 2005). The

gills of suspension-feeding bivalves are organs with a large

surface that create water currents by moving their ciliated

surface. M. falcata gills consist of two ctenidia, each formed by

two V-shaped demibranchs and the latter are composed by a

group of numerous gill filaments that can be divide into frontal

zone, intermediate zone and abfrontal zone (David and

Fontanetti, 2005).

According to Dumouhtsidou and Dimitriadis (2004), M.

galloprovincialis gill filament consists mainly of a single

layer of various types of epithelial cells (ciliated and non-

ciliated columnar cells and mucous cells) and endothelial

cells surrounding a central lumen and resting on a basement

membrane. Gomez-Mendikute et al. (2005) described, in the

same species, the cell types present in gill filaments, as

follows: in the frontal zone five cell types (columnar frontal

cells with short cilia, large eu-latero-frontal cells with cirri,

post-latero-frontal cells with no cilia, large lateral cells with

www.elsevier.com/locate/micron

Micron 39 (2008) 329–336

* Corresponding author. Tel.: +55 19 35264135; fax: +55 19 35264136.

E-mail address: joseaugustodavid@hotmail.com (J.A. de Oliveira David).

0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2007.06.002

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large nuclei and long cilia and small non-ciliated post-lateral

cells); flattened endothelial cells in the intermediate zone and

ciliated and non-ciliated cells in the abfrontal zone.

Mucocytes were also identified by those authors in the

frontal and abfrontal zones, as well as lipofuscin granules in

cells of the three zones.

Due to the importance of this organ to the bivalve mollusks

and the fact that gills have a large surface constantly exposed to

water and its substances (harmful or not), the gill filaments

morphology has been extensively used as an indicator of

aquatic pollution (Gregory et al., 2002). According to Sunila

(1988), because of their simple epithelium with very

specialized cells, gill filaments are suitable for histopatholo-

gical analysis, in which the effects of water-soluble pollutants

are easily observed.

In this study, M. falcata gill filaments were characterized

for the first time using histology, histochemistry and

transmission electron microscopy to provide the framework

for future histopathological studies to detect the effect of

pollutants in the gills of this bivalve broadly distributed in

Brazilian estuaries.

2. Materials and methods

Specimens of M. falcata with approximately 4.0 cm were

collected in Santos estuary (Brazil) (23855.0520S;

46826.9750W) and transported to the laboratory, where they

were maintained in 5 L aquaria containing water of the

collecting site with constant aeration. The area where the

specimens were collected is not under the influence of

industries and can be considered as an unpolluted site. Small

gill fragments from 19 individuals were excised and fixed for

histological and histochemical analysis; for the ultrastructural

analysis gill fragments from 10 individuals were used.

Fig. 1. Histological sections of Mytella falcata gill filaments (H/E). (A) General view of gill filaments with ciliary discs; (B) view of a gill filament showing its zones

and the haemolymph vessel; (C) detail of the haemolymph vessel and the abfrontal zone; (D) detail of the frontal zone of the filament. Abbreviations: az, abfrontal

zone; cd, ciliary discs; ed, endothelium; ep, epithelium; fc, frontal cells; fz, frontal zone; g, granulocyte; iz, intermediate zone; lc, lateral cells; lfc, eu-latero-frontal

cells; v = haemolymph vessel. Scale bar in A = 20 mm and in B, C and D = 10 mm.

J.A. de Oliveira David et al. / Micron 39 (2008) 329–336330

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2.1. Histology

Gill fragments were fixed in 4% paraformaldehyde in 0.1 M

phosphate buffer solution pH 7.4. The material was submitted

to the same buffer for 24 h and dehydrated in an ascending

series of ethanol baths. The material was then embedded (24 h)

in JB4 historesin with a catalyst at 4 8C to avoid premature

polymerization and placed in an incubator at 37 8C for

polymerization. Blocs were sectioned with 5 mm using the

Sorvall JB4 (Bio Rad) microtome with glass knives. Sections

were stained with haematoxylin and eosin (H/E) according to

histology routine.

2.2. Histochemistry

The material fixed in aqueous Bouin solution, was processed

according to the same histological procedures described

previously; however, to remove the picric acid residues, the

material was washed four times (5 min each) with phosphate

buffer pH 7.4 prior to the dehydration. Sections were submitted

to histochemical tests for detection of polysaccharides—PAS

(Junqueira and Junqueira, 1983), total proteins—bromophenol

blue (Pearse, 1985) and collagen—picrosirius-hematoxylin

(Junqueira and Junqueira, 1983) with some modifications, as

follows: the sections were rehydrated in water for 1 min and

submitted to the picrosirius solution at 60 8C for 60 min in an

incubator at 60 8C, the slides were washed in water, counter-

stained with hematoxylin for 5 min, washed in water for

15 min, air dried and mounted with Canada balsam.

2.3. Transmission electron microscopy

Small gill fragments were fixed in 2.5% glutaraldehyde in

0.1 M cacodylate buffer pH 7.2 at 4 8C and post-fixed in 1%

osmium tetroxide for 2 h. The material was contrasted with 2%

uranyl acetate in 10% ethanol for 4 h, dehydrated in a ascending

series of acetone, embedded in Epon-araldite with catalyst for

24 h and then placed in an incubator at 70 8C for 24 h. Ultrathin

sections were, placed in grids and the material was examined

and photographed using a transmission electron microscope

(TEM) Phillips CM 100.

3. Results

Ciliary discs composed of ciliated columnar cells inter-

connect gill filaments of M. falcata. Cilia from cells of a

filament intercalate between cilia from cells of the following

filament, thus connecting each other (Fig. 1A). The filament

may be divided into frontal, intermediate and abfrontal zones

(Fig. 1B). The frontal zone differs from other zones by the

presence of ciliated cells (Fig. 1B and D). A haemolymph

vessel, located in the central portion of the filament (Fig. 1A–

C), is internally lined by an endothelium composed of cells with

very flat nucleus. In these cells, part of the chromatin is

condensed and mainly located in the periphery of the nucleus.

Also, in the cytoplasm, mitochondria and a well-developed

Golgi complex are observed (Fig. 2A).

The intermediate zone consists of a simple squamous

epithelium (Fig. 1C) formed by epithelial cells exhibiting a flat

nucleus, abundant microvilli in their apical surface (Fig. 2B)

and many vesicles near the surface of cells (arrowhead in

Fig. 2. Electron micrographs of the intermediate zone of Mytella falcata gill

filament. (A) Endothelial cell; (B) epithelial cell; detail, vesicle; (C) detail of

junctions observed in all cells forming the gill surface. Abbreviations: aj,

adhesive junction; ct, connective tissue; ed, endothelium; ep, epithelium; gc,

Golgi complex; mv, microvilli; n, nucleus; rer, rough endoplasmic reticulum; sj,

septate junction; v, haemolymph vessel; arrowheads, vesicle. Scale bar in A and

B = 3 mm and in the detail and C = 0.3 mm.

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Fig. 2B and in detail). Adhesive junctions and septate junctions

connect all cells lining gill filaments (Fig. 2C).

Between the epithelium and endothelium, there is a structure

that gives support to the haemolymph vessel and the gills. This

structure is stained with PAS (asterisk in Fig. 3A) and

picrossirius (asterisk in Fig. 3D), indicating the presence of

collagen and polysaccharides, and under TEM, this structure

exhibits fibrous elements (Fig. 2A and B). Due to its

composition this structure will be called in this study as

‘‘connective tissue’’ as discussed latter.

Inside the haemolymph vessel, haemocytes are commonly

seen and may be distinguished between granulocytes and

agranulocytes. Granulocytes are strongly stained by eosin

(Fig. 1C) and bromophenol blue (Fig. 3B), demonstrating the

presence of protein granules in the cytoplasm. Agranulocytes

are weakly stained by eosin and bromophenol blue (Fig. 3B).

Fig. 3. Mytella falcata gill filaments stained with PAS (A), bromophenol blue (B and C) and pricrosirius-haematoxylin (D). (A) General view of gill filaments

showing the position of mucous cells (strongly stained) and the connective tissue; (B) note the prominent staining for proteins in the region of basal bodies and

granulocytes, and weak stained agranulocytes; (C) detail of the frontal zone showing a secretory cell with granules stained with bromophenol blue; (D) note the

presence of collagen (red staining) in the connective tissue. Abbreviations: a, agranulocyte; az, abfrontal zone; fz, frontal zone; g, granulocyte; iz, intermediate zone;

v, haemolymph vessel; *, connective tissue; arrows, secretory cells of the frontal zone; arrowhead, basal bodies. Scale bars = 30 mm.

J.A. de Oliveira David et al. / Micron 39 (2008) 329–336332

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Under TEM, granulocytes present many digestive vacuoles

with myelin figures (Fig. 4A). Haemocytes are able to cross the

connective tissue (Fig. 4A) and reach the gill epithelium in any

part of the filament. Nerve bundles (Fig. 4B) are present near

the connective tissue, especially in the abfrontal and frontal

zones of the filament.

The abfrontal zone is formed by cuboidal cells, which

exhibit round nucleus (Fig. 1C), several microvilli in the

surface and few cilia (Fig. 4C); vesicles similar to those found

in epithelial cells from the intermediate zone are also observed

(arrowhead in Fig. 4C). Mucous cells strongly stained with PAS

are very common in the abfrontal zone and are also observed in

the frontal near the intermediate zone (Fig. 3A); these cells

contain granules of different sizes and electron densities

(Fig. 4C).

The frontal zone is composed of five cell types; two are

devoid of cilia (absorptive cell and secretory cell), while the

remaining three can be distinguished by the type of cilia (frontal

cell, eu-latero-frontal cell and lateral cell) (Fig. 1D).

Ciliated cells are columnar and exhibit a large number of

mitochondria immediately beneath the basal bodies (Fig. 5A–

C), this region is easily observed when stained with

bromophenol blue (arrowhead in Fig. 3B). Ciliary rootlets

are present among mitochondria. The rootlets are striated

structures that extend from the basal bodies until the basal

portion of the cell. In all ciliated cells, the endoplasmic

reticulum and the Golgi complex are also observed; the nucleus

(Fig. 5A and C) is large and round shaped. The Golgi complex

and the rough endoplasmic reticulum of eu-latero-frontal and

frontal cells are more developed compared to those of lateral

cells. On the other hand, the latter exhibit larger and more

abundant mitochondria in comparison with other cells

(Fig. 5B). All cells of the frontal zone exhibit several

microvilli on the surface and in the cytoplasm, multivesicular

bodies and digestive vacuoles (Fig. 5) are present, these

structures are observed in lower numbers in lateral cells

(Fig. 5B).

Absorptive cells (Fig. 5D) lack cilia, their free surface is

covered by several microvilli and many pinocytic vesicles are

present in the apical region, these vesicles are formed in the

base of microvilli (arrows in Fig. 5D). The nucleus is elongated

with uncondensed chromatin. These cells are located between

lateral cells and eu-latero-frontal cells.

Secretory cells are located in between frontal cells and are

rarely observed. PAS (arrow in Fig. 3A) as well as bromophenol

blue (arrow in Fig. 3C) staining is observed in these cells,

suggesting a glycoprotein secretion, unlike the secretion of

mucous cells.

4. Discussion

M. falcata gill filaments were examined by Narchi and

Galvao-Bueno (1983) at the light microscopy level; these

authors described the general morphology of the filament

without detailing the structure of its cells. David and Fontanetti

(2005) studied the morphology of the gill surface of this species

at the scanning microscopy level and described the cilia

Fig. 4. Electron micrographs of Mytella falcata gill filament. (A) Detail of a

granulocyte crossing the connective tissue; (B) detail of a nerve bundle; (C)

detail of the abfrontal zone showing a mucous cell. Abbreviations: ct, con-

nective tissue; dv, digestive vacuole; ep, epithelium; g, granulocyte; mc, mucous

cell; mv, microvilli; n, nucleus; nb, nerve bundle; v, haemolymph vessel; arrow,

cilium; arrowheads, vesicle. Scale bar in A and B = 1 mm and in C = 3 mm.

J.A. de Oliveira David et al. / Micron 39 (2008) 329–336 333

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distribution along the filament. The present study is the first to

use histochemical and ultrastructural techniques to analyze the

gill filaments of M. falcata.

The shape and distribution of cilia present in the frontal

zone, observed in this study, are very similar to those observed

in other species. These cilia have an essential role in capturing

and transporting particles during feeding and, therefore, are

apparently highly conserved in the group (Owen, 1974;

Jorgensen, 1996; Gregory and George, 2000).

The ciliary movement is ATP-dependent and mitochondria

are the organelles capable of converting energy from nutrients

into ATP, providing the energy source necessary to this

process. In this study, larger and more abundant mitochondria

were observed in lateral cells. This might be associated with

their role in the surface of the gill filament. According to Owen

(1974), lateral cells are mainly responsible for water

circulation inside the bivalve body, causing its passage

between two filaments. On the other hand, Jones et al.

(1990) suggests that this role, in Mytilus edulis, may be divided

with cilia found in the abfrontal surface. When comparing the

abfrontal ciliation of M. falcata and other bivalvia species, it

was concluded that M. falcata abfrontal cilia were not detected

Fig. 5. Electron micrographs of cells in the frontal zone of Mytella falcata gill filament. (A) Eu-latero-frontal cells; (B) lateral cell; (C) frontal cell; (D) absorptive

cell. Abbreviations: bb, basal body; gc, Golgi complex; m, mitochondria; mv, microvilli; n, nucleus; pv = pinocytic vesicles; rer, rough endoplasmic reticulum; *,

multivesicular bodies and digestive vacuoles; arrow, formation of pinocytic vesicles. Scale bars = 3 mm.

J.A. de Oliveira David et al. / Micron 39 (2008) 329–336334

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in numbers high enough to generate water circulation inside

the bivalve body. This idea agrees with the study of David and

Fontanetti (2005) that concluded that M. falcata abfrontal

ciliation was similar to that found in Modiolus modiolus by

Dufour and Beninger (2001) and was not sufficient to promote

water circulation.

The cytoplasm of lateral cells exhibited the least amount of

organelles, except by mitochondria, which are present in large

numbers. On the surface of these cells, few microvilli were

observed. This might be associated with the high specialization

of these cells, which play a major role in water circulation

inside the body of the organism and thus are vital for the

animals survival. Other cell types, in the frontal as well as other

zones of the filament, exhibit a cytoplasm with small pinocytic

vesicles that reflect the absorptive nature of the entire gill

epithelium. This fact is in agreement with Junqueira and

Carneiro (2005) that suggested that the role of microvilli might

be associated with the absorption of substances from the

environment into cells, as they increase surface area. Fawcett

and Porter (1954) discussed the presence and role of microvilli

on cells from the frontal surface of gill filaments and concluded

that microvilli might function as a mechanical support for an

amorphous mucous material and together, microvilli and

amorphous material, would strengthen the anchorage of cilia.

In this study, a moderate PAS staining was observed in the

surface of gill epithelium (Fig. 4A) suggesting the presence of a

polysaccharide layer that may act as a protection against

potentially harmful agents, as well as a connecting point for

particles to be ingested.

The absorptive cell between frontal cells and eu-latero-

frontal cells was called by some authors as post-latero-frontal

cell and considered without a known function (Gregory et al.,

2002; Gomez-Mendikute et al., 2005). Some authors suggested

that the absence of ciliation in these cells was important to crate

a space where lateral cilia and eu-latero-frontal cilia could carry

through their movement (Aiello and Sleigh, 1972). In the

present study, the term absorptive cell was used because this

cell presented many microvilli and pinocytic vesicles suggest-

ing material uptake from the environment.

In epithelial and abfrontal cells, the vesicles near the cell

membrane have a specific role that still needs clarification.

However, the content of these vesicles presents more electron

density when compared to mucous cells granules, excluding the

idea of a small secreting mucous vesicle.

The junctions that connect the different cell types of gill

filaments of M. falcata are structures commonly found in

invertebrates with specific roles and morphological character-

istics. The adhesive junction located in the apical portion of

cells form a ring that encircles the entire cell and acts as a

connecting point to the cytoskeleton network, increasing the a

stiffness of the brush border. The septate junctions also form a

ring around the entire cell and play a role in occlusion, not

allowing the passage of material from the environment through

intercellular spaces, and in communication and adhesion

among cells of the tissue (Satir and Gilula, 1970).

The structure supporting the haemolymph vessel has been

observed by other authors, but its constitution is not well

defined. Most authors regard it as a chitinous-like structure

(Gregory et al., 2002), but others name the structure as a

basement membrane (Dumouhtsidou and Dimitriadis, 2004). In

the present study, the characteristics observed with PAS and

picrosirius staining and the ultrastructural analysis, demon-

strated that this structure exhibits polysaccharides associated

with collagen. It was also observed that haemocytes are capable

of cross this structure and since haemocytes are not able to cross

chitinous-like layers, the term connective tissue was applied in

this study. Thus this study classified the structure as an acellular

connective tissue rich in polysaccharides and collagen that

gives sustentation to gill and the haemolymph vessel. The

thickness of this connective tissue is variable according to

friction that is subjected to gill filaments, caused by constantly

water circulation in the interfilamentar space; this is observed in

the ciliary discs region where the connective tissue is thicker

(Fig. 1A).

Acknowledgements

The authors are thankful to CAPES, CNPq and FUNDU-

NESP for financial support, to ‘‘Nautica da Ilha’’ and Marcia

M. Hoshina for helping during collecting trips, to Cristiane M.

Mileo, for the illustrations, and to Gerson Mello Souza, Monika

Iamonte and Antonio T. Yabuki for the support during technical

procedures.

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