Chalcedony (a crystalline variety of silica): Biogenic origin in electric

organs from living Psammobatis extenta (family Rajidae)§

Marıa Prado Figueroa a,*, Facundo Barrera a, Nora N. Cesaretti b

a Instituto de Investigaciones Bioquımicas de Bahıa Blanca (INIBIBB), CONICET/UNS, Department de Biologıa, Bioquımica y Farmacia,

Universidad Nacional del Sur, La Carrindanga Km 7, C.C. 857, FWB8000 Bahıa Blanca, Argentinab Department de Geologıa, Universidad Nacional del Sur, San Juan 670, 8000 Bahıa Blanca, Argentina

Received 26 April 2007; received in revised form 14 August 2007; accepted 14 August 2007

Abstract

The electric organs of electric fish have been used extensively for the study of peripheral cholinergic synapses. Aluminum and silicon have been

observed in the electrocytes of Psammobatis extenta, a fish belonging to the family Rajidae, using a combination of scanning electron microscopy

and X-ray spectrometry. Based on this evidence, the presence of silica minerals has been documented by means of mineralogical techniques.

Electric organ cryostat sections and subcellular fractions were observed using a Leica DMLP mineralogical microscope. The shape, size and color,

among other properties, were analyzed in plane-polarized light, while birefringence and the extinction angle, which allow for mineral

identification, were observed through crossed-polarized illumination. The distribution of chalcedony, an oxide silicon mineral, in the sections

and all the fractions of the electric organ was recorded. X-ray diffraction analysis of the electric organ segments showed a similar result, with a low-

quartz variety. Chalcedony precipitation occurred at a specific pH (7–8) and oxidation potential (Eh; 0.0 to �0.2). This observation supports the

important role played by pH and Eh conditions in silica precipitation in electrocytes, as has been reported in geological environments. It is possible

that silica formation and silica degradation in electric organs are also related to the enzymes, silicatein and silicase, that direct the polymerization

and depolymerization of amorphous silica in sponges. Carbonic anhydrases (silicase) are involved in physiological pH regulation. Crystallization

of chalcedony via spiral growth from a partially polymerized fluid is consistent with processes known to occur in organic systems. This is the first

time that a biogenically produced crystalline mineral phase (i.e., chalcedony) has been observed in the electrocytes and cholinergic nerves from

living electric fish.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: AChR; Biosilicification; Carbonic anhydrases; Electric fish; Neurodegeneration; Permineralization; Silicase; Silicatein; Silicon; Taphonomy

www.elsevier.com/locate/micron

Micron 39 (2008) 1027–1035

1. Introduction

Electric organs have been used for the study of peripheral

cholinergic synapses. The electric organs from the family

Rajidae are derived from skeletal myoblasts of the caudal

region and the electrocytes, electric organ cells, have different

shapes (Fessard, 1958). Electrocytes from Psammobatis

extenta are cup-shaped cells, highly polarized, with an anterior,

concave, innervated face and a posterior, convex, non-

innervated face, that shows a very large system of caveolae

§ This work was presented as an abstract in December 2005 (Prado Figueroa

et al., 2005).

* Corresponding author. Tel.: +54 291 4861201x122; fax: +54 291 4861200.

E-mail addresses: mpfigueroa@uns.edu.ar, inprado@criba.edu.ar

(M. Prado Figueroa).

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

doi:10.1016/j.micron.2007.08.004

(Prado Figueroa et al., 1995). Neuronal cell death and synaptic

terminal degeneration have been noted in the adult electric

organs of fish from the family Rajidae (Fox et al., 1990 and our

observations). Based on the evidence of aluminum and silicon

accumulation in electrocytes from P. extenta by scanning

electron microscopy and energy dispersing X-ray microana-

lysis (SEM-EDS; Barrera et al., 2001; Prado Figueroa et al.,

2003; Prado Figueroa and Santiago, 2004; Prado Figueroa and

Santiago Contreras, 2007), we documented the presence of

silicon minerals. It was thought that these compounds could

form minerals (i.e., solid inorganic substances with a defined

chemical composition and determined crystallography). In

neurodegenerative diseases and in normal elderly brains,

aluminum, iron and silicon have been observed within the

neuron (Perl and Brody, 1980; Candy et al., 1985; Honda et al.,

2004; Huang et al., 2004; Domingo, 2006). Data obtained by

M. Prado Figueroa et al. / Micron 39 (2008) 1027–10351028

SEM-EDS, mineralogical microscopy and X-ray diffraction on

the electric organs of P. extenta obtained from the Bahıa Blanca

Estuary and the San Matıas Gulf, Argentina, are reported

herein.

2. Materials and methods

2.1. Specimens

Adult female and male P. extenta were collected from the

Bahıa Blanca Estuary (388400S and 398300S, 628160W and

638260W) in the Buenos Aires Province of Argentina and

transported to the laboratory in sealed polyethylene bags

containing oxygen-saturated seawater. The fish were

anesthetized by immersion in ice cold seawater for 10 min

and then killed by pithing. Immediately after dissection of

the ray, the electric tissue was frozen in liquid nitrogen at

�198 8C. Adult fish were also caught from the San Matıas

Gulf (408350S and 428150S, 658150W and 638300W) in the Rıo

Negro Province of Argentina. Frozen fish were then

transported to the laboratory where they were preserved

and refrigerated at �80 8C. For these experiments, 10

samples were analyzed. Fish were collected at different

seasons during the last 10 years (Prado Figueroa et al., 1995;

Vidal et al., 1997; Barrera et al., 2001; Prado Figueroa et al.,

2003; Prado Figueroa and Santiago, 2004; Prado Figueroa

and Santiago Contreras, 2007).

2.2. SEM-EDS

An electric organ cytoplasmic extract (50 ml) on lyophilised

paper (Labconco Corp., USA) was fixed in 2.5% glutaralde-

hyde in a 0.05 M sodium phosphate buffer (pH 7.2) for 60 min

at 4 8C. Samples were washed with buffer and bi-distilled water

for 2 h, then, dehydrated in series with 50, 75 and 100% ethanol

and 100% acetone (10 min in each solution) and air-dried.

Samples were subsequently metallized with gold (200 A) or

carbon in a sputter coater model 3 (Pelco, Ted Pella Inc., Tustin,

CA, USA). The samples were then mounted on lyophilised

paper with adhesive paper on a bronze support. Lastly, the

samples were oriented for observation through a JEOL 35

scanning electron microscope at 15 kV, equipped with an

EDAX Si(Li) energy dispersive X-ray detector, from the Centro

Regional de Investigaciones Basicas y Aplicadas de Bahıa

Blanca (CRIBABB).

2.3. Cryostat sections

Cryostat sections, approximately 7–8 mm in thickness, of

the electric organs were performed along the antero-posterior

axis (model CTI; International Cryostat IEC, USA) at �20 8C.

Sections were then collected on glass slides and fixed with 3.7%

formaldehyde in 0.1 M PBS (pH 7.4) for 20 min, washed with

PBS and mounted with PBS/glycerol (1:1). Thin sections were

observed and photographed using plane-polarized light and

crossed-polarized illumination with a mineralogical Leica

Microscope DMLP, at 10, 25 and 40�.

2.4. Electric organ fractionation

Fractionation of electric tissue homogenates by differential

centrifugation was carried out as described for other tissues

(Beaufay and Amar-Costesec, 1976; Amar-Costesec et al.,

1985) using isotonic 3 mM imidazole–HCl-buffered 0.25 M

sucrose (pH 7.4). The following fractions were obtained:

cytoplasmic extract (E), nuclear fraction (N), large granules

(ML), microsomes (P) and supernatant (S). Drops of the

fractions were collected on glass slides, dried and mounted in

PBS/glycerol. These fractions were inspected using a miner-

alogical microscope under similar conditions to those used for

thin sections.

2.5. Mineralogy

The DMLP polarized light microscope (mineralogical)

used for the present study has a polarizer and a switchable

analyzer. In mineralogical microscopy, when the light enters

an anisotropic mineral, one which transmits light at different

rates in different orientations, it is decomposed in two rays,

oscillating in two orthogonal planes. This phenomenon is

known as birefringence and allows for the identification of

each mineral. In this microscope, with a circular graduated

stage capable of a 3608 rotation, the minerals in different

positions display their optical properties, such as birefrin-

gence color and extinction position, with crossed polarizers.

This represents the essential difference from a biological

microscope.

2.6. X-ray diffraction

A Rigaku-Denki equipment for X-ray determinations

(Geology Department, UNS) was used for analyzing the

structural mineral present in the electrocytes. The operation

conditions were 35 kV, 15 mA, Cu/K-alpha 1 and a Ni

filter.

Electric organs without any treatment were used for X-ray

diffraction analysis. The sample was dried at 60 8C, milled with

an agate mortar and then mounted with adhesive on a

diffraction support.

3. Results

3.1. SEM-EDS

An electric organ cytoplasmic extract metallized with gold

was microanalized in Fig. 1. The energy dispersive spectrum

(Fig. 1a) showed high peaks at energy lines characteristic of

oxygen, silicon and aluminum. Sodium, potassium, calcium

and zinc were also observed. Weight and atomic percents for

these elements are indicated in Fig. 1b.

Simultaneous scanning electron micrograph of the micro-

analized electric organ cytoplasmic extract showed a granular

structure (Fig. 1c). Many organelles were 1–2 mm in size. X-ray

EDS maps were also obtained. Zinc, oxygen and silicon maps

are shown (Fig. 1d–f).

Fig. 1. Electric organ cytoplasmic extracts on lyophilised paper and metalized with gold were microanalized. This energy dispersive spectrum (a) shows high peaks

localized at energy lines characteristic for oxygen, silicon and aluminum. Sodium, potassium, calcium and zinc are also observed. Weight and atomic percents for

these elements are indicated next to the spectrum (b). The measurement was based on the dimension of the area shown in the SEM micrographs (c). Simultaneous

scanning electron micrograph of the electric organ cytoplasmic extract microanalized shows a granular structure (c). Many organelles are about 1–2 mm in size. X-ray

distribution maps for zinc, oxygen and silicon are shown (d–f).

M. Prado Figueroa et al. / Micron 39 (2008) 1027–1035 1029

3.2. Cryostat sections

Photomicrographs of cryostat sections from the electric

organ of P. extenta are shown in Figs. 2 and 3. Sections

illuminated with a plane-polarized light only showed the

biological aspects and the mineral was uncolored, thus virtually

invisible and having the same refractive index as the tissue.

These electrocytes were semi-circular in shape with their

Figs. 2 and 3. (2) Photomicrographs of cryostat sections from electric organs of P. extenta. (a) Sections illuminated with a plane-polarized light show only the

biological aspects, while the mineral is uncolored. Electrocytes from P. extenta are semi-circular in shape with their concave face, innervated (IF); the non-innervated

face (NIF) is convex and posterior. (b) The crossed-polarizer image shows SiO2 replacements in grey and white arrangements. Chalcedony is distributed in the

electrocytes (partial silicification, white arrows) and along the curved nerves (full silicification, black arrow). (3) Photomicrographs at high magnification with

crossed-polarizers (a and b) show a silicified, concave, innervated region (black arrows). Silicification in the electrocyte (white arrows).

M. Prado Figueroa et al. / Micron 39 (2008) 1027–10351030

concave face innervated (IF); the non-innervated face (NIF)

was convex and posterior (Fig. 2a). The crossed-polarizer

image showed chalcedony, a silicon mineral, with a first-order

birefringence color (pale grey-to-white), filled and correspond-

ing to the shape of the electrocyte. Chalcedony is a SiO2 variety

mineral, its extinction is undulatory and it is distributed in the

electrocytes (Fig. 2b, white arrows) and nerves. Full silicifica-

tion was shown in some nerves (Fig. 2b, black arrow).

Photomicrographs at high magnification with a crossed-

polarized light showed partial silicification of the innervated

region (Fig. 3a and b, black arrows). Silicifications in the

electrocyte (Fig. 3b, white arrows).

3.3. Electric organ fractions

Fractionation of electric tissue homogenates by differential

centrifugation was carried out using isotonic 3 mM imidazole–

HCl-buffered 0.25 M sucrose (pH 7.4). Photomicrographs of all

fractions: cytoplasmic extract (E), nuclear fraction (N), large

granules (ML), microsomes (P) and supernatant (S) from the

M. Prado Figueroa et al. / Micron 39 (2008) 1027–1035 1031

electric organs of P. extenta are shown in Fig. 4a and b (E),

Fig. 5a and b (N), Fig. 6a and b (ML), Fig. 7a and b (P), Fig. 8a

and b (S) and Fig. 9a and b (also S); with plane-polarized light

(in ‘‘a’’ parts) and crossed-polarized illumination (in ‘‘b’’

parts). All ‘‘b’’ parts photomicrographs show SiO2 replace-

Figs. 4–9. (4) Photomicrographs of a cytoplasmic extract (E). (a) In plane-polarize

crossed-polarizers, the presence of silica following the previous morphology. All (b)

white arrangements (chalcedony). (5) Photomicrographs of a nuclear fraction (N). (

silica replacement. (6) Photomicrographs of large granules (ML). (a) Plane-polarized

(7) Photomicrographs of microsomes (P). (a) Plane-polarized light shows red dendrit

of the supernatant (S). (a) Plane-polarized light shows isolated particles; (b) crossed-p

the supernatant (S; detail of Fig. 8). (a) Plane-polarized light shows a pale red mi

ments in grey and white arrangements (chalcedony) with

undulatory extinction. Fig. 9a and b of the supernatant fraction

(S) show a pale red mineral in plane-polarized light, which

exhibited the same birefringence with crossed-polarizers as in

Fig. 8a and b.

d light, the red color in this fraction may indicate the presence of Fe3+; (b) in

photographs are with crossed-polarizers showing SiO2 replacements in grey and

a) Plane-polarized light shows the dendritic shape; (b) crossed-polarizers show

light shows an irregular shape; (b) crossed-polarizers show silica replacement.

ic aspect; (b) crossed-polarizers show silica replacement. (8) Photomicrographs

olarizers show replacement of some particles by silica. (9) Photomicrographs of

neral; (b) crossed-polarizers show replacement of some particles by silica.

Fig. 10. X-ray diffractometric analysis. A spectrum with different peaks was obtained from this analysis. The peaks, 4.2519, 3.3488, 2.4611 and 2.2829 A, belong to

an a quartz (low quartz). The 2.36 A peak may belong to some zinc mineral.

M. Prado Figueroa et al. / Micron 39 (2008) 1027–10351032

3.4. X-ray diffractions

Different peaks were obtained from diffractometric analysis

(Fig. 10); specifically, 4.2519, 3.3488, 2.4611 and 2.2829 A

peaks belonged to the a quartz (low quartz; Moore and

Reynolds, 1997). The 2.36 A peak may have belonged to some

zinc mineral; this element was also detected by SEM-EDS

(Fig. 1).

4. Discussion

Neuronal cell death and synaptic terminal degeneration have

been described in adult skate electric organs (Fox et al., 1990;

our observations). Understanding cellular and molecular

mechanisms participating in neurodegenerative processes is

thus an important field of research. The spatial resolution of

SEM-EDS is an analytical technique combining simultaneous

compositional and morphological observations (Morgan et al.,

1999). Energy-dispersive spectra of the electrocytes of P.

extenta show peaks localized at energy lines, characteristic of

oxygen and aluminum (Barrera et al., 2001; Prado Figueroa

et al., 2003; Prado Figueroa and Santiago, 2004). In this study,

the spectra also showed peaks for aluminum, silicon and zinc.

Mineralogical microscopy allows for identification of

cellular lesions and their mineralization not visible through

biological microscopy. Chalcedony, a crystalline variety of

SiO2, was identified in the cytoplasm and synaptic regions of

electrocytes when observed with a mineralogical microscope.

Prado Figueroa et al. (2005) and Prado Figueroa and Cesaretti

(2006) documented, for the first time, the presence of silica in

cryostatic sections as well as in the subcellular fractions of

electric organs of P. extenta during oxidative stress.

The SiO2 mineralization can occur under a wide variety of

circumstances and the origin of chalcedony is widely debated in

the literature (Heaney, 1993; Fernandez Lopez, 2000; Nash and

Hopkinson, 2004).

Alpha quartz or low quartz, the crystalline variety of silica

found in this study, is the most common of silicon minerals in

sedimentary rocks (Moore and Reynolds, 1997). A slight over-

saturation of silicon is necessary for allowing chalcedony

precipitation from the solution. Chalcedony seems to form and

persist below 180 8C (Fournier, 1985); it is a cryptocrystalline

variety of compact silica which contains little quartz crystals

with submicroscopic pores (Deer et al., 1966). Chalcedony is

not simply fine-grained quartz (Heaney et al., 1994).

Chalcedony is a microcrystalline fibrous form of silica which

can occur in optically length-fast or length-slow forms, but

actually consists of nanoscale intergrowths of quartz and the

optically length-slow fibrous silica polymorph moganite; these

are both silica minerals, but they differ in that quartz has a

Fig. 11. Diagrams: (a) solubility of silica and alumina as a function of pH (Mason, 1956) and (b) Eh and pH diagram (Krumbein and Garrels, 1952).

M. Prado Figueroa et al. / Micron 39 (2008) 1027–1035 1033

trigonal crystal structure, whilst moganite is monoclinic.

(Conrad et al., 2007; Heaney and Post, 1992; Heaney, 1993;

Heaney et al., 2007).

The relationship between pH and Eh, and the solubilities of

silica, is well-known. SiO2 and alumina precipitation occur at

an acidic pH when the system is inorganic (Fig. 11a; Mason,

1956). When organic matter and inorganic substances co-exist,

the pH conditions for silica precipitation change. The

conditions at which silica precipitation occur is at a pH 7 or

near a pH of 8 and an Eh (oxidation potentials) of 0.0 to �0.2

(Fig. 11b; Krumbein and Garrels, 1952).

It was found that the common marine sponge, Tethya

aurantia, produces large quantities of glass-like silica needles;

these are so abundant that they represent 75% of the dry weight

of the sponge (Shimizu et al., 1998). Recent evidence shows

that the formation of spicules is mediated by the enzyme,

silicatein (Cha et al., 1999). It has been observed that both iron

and silicate stimulate the activity of silicatein (Muller et al.,

2003). Silicatein also has proteolytic (cathepsin-like) activity

(Shimizu et al., 1998; Muller et al., 2003). In organic systems,

this is consistent with the crystallization of chalcedony from a

partially polymerized fluid (Heaney, 1993). In a marine sponge,

these fluids are polymerized by silicatein (Cha et al., 1999).

Iron is also necessary for silica precipitation in a geological

environment (Heaney, 1993) and, in our samples, iron was

replaced by chalcedony (Fig. 9a and b). In these figures, the pale

red mineral was interpreted to be an iron mineral which was

partially replaced by chalcedony. From different spectra

obtained by EDS-SEM, iron was only recorded in some (not

shown herein). Prado Figueroa (2005) documented oxidative

stress in electric organs from family Rajidae. In such oxidative

conditions, the presence of iron could contribute to silica

formation.

The observations with the mineralogical microscope may

have shown the initial process of oxidation (iron mineral) and

the end of this process by silicified mineral. It seems that

initially silica would be in a soluble phase, at a pH > 8 and an

Eh > 0.0. Under these conditions, iron could exist as Fe3+

(Fig. 11b). If changes in pH and Eh values occurred (Fig. 11b),

for example, a decrease of pH to 7–8 and an Eh to 0.0 to (�0.2),

this combination could result in silica precipitation. The

common association between length-fast chalcedony and ferric

iron oxyhydroxides suggests that chalcedony crystallization is

favoured where catalysis by ferric ion can occur (Hopkinson

et al., 1999). It may be possible to consider a wider range of Eh

in biological systems, extending to more positive values of Eh

and giving an evolution for reaching SiO2 conditions for

precipitation in the presence of Fe3+. In Fig. 4a and b, it was

possible to distinguish a red color in the cytoplasmic extract,

which could be attributed to the presence of Fe3+. Then, this

could serve as the basis of crystalline variety precipitation.

On the other hand, oxidative damage in the brain is often

associated with iron which has pro-oxidative properties (Honda

et al., 2004; Mura et al., 2006). Iron-mediated oxidative damage

in the brain is compounded by the fact that the distribution of

iron in the brain is non-uniform, being particularly high in areas

sensitive to neurodegeneration (Mura et al., 2006).

An important role is played by SiO2 accumulation, pH and

Eh, which suggests that the electrocytes are being replaced by

SiO2 and, according to a taphonomic point of view, are

becoming mineralized (Efremov, 1940). This mineralization or

cementation (inorganic addition by precipitation into an open

space or replacement of previous organic material) of the

electrocytes implies the death of the cell and the nerves,

revealing that these conditions of pH and Eh are necessary for

this process to occur.

From a biological point of view, silica formation in electric

organs may be mediated by the enzyme silicatein, as reported

by Shimizu et al. (1998) and Cha et al. (1999) in the sponge. On

the other hand, silica precipitation in biological environments

could indicate a permineralization process; as soft-tissue

becomes permeable, the fluid circulations can be placed and the

mineral precipitation (formation) can occur (Fernandez Lopez,

2000).

It was thought that this process could also occur in the

human brain due to the similarities in the occurrence of Si and

M. Prado Figueroa et al. / Micron 39 (2008) 1027–10351034

Al in both systems (Perl and Brody, 1980; Candy et al., 1985;

Fasman et al., 1995; Domingo, 2006). This was corroborated by

preliminary studies in the human brain by mineralogical

microscopy where chalcedony was found (Prado Figueroa

et al., 2006). In addition, in some fractions (S) of the electric

organs, zinc is one of the observed elements, as well as being

identified in the cytoplasmic extract by SEM-EDS. This is

similar to the Huang et al. (2004) studies where Cu, Fe and Zn

enrichment were associated with neurodegenerative diseases in

the human brain. Valko et al. (2005) attributed a neurodegen-

erative role to the presence of Zn.

Silicase, a member of the family of carbonic anhydrases, is

able to depolymerize amorphous silica in sponges (Schroder

et al., 2003). Carbonic anhydrases (CAs) are a family of zinc

metalloenzymes (Sly and Hu, 1995). CAs are physiologically

important enzymes that catalyze a reversible conversion of

carbon dioxide to bicarbonate and participate in ion transport

and pH control (see Schroder et al., 2007). CAs may play an

important role in human brain (Fedirko et al., 2007; Kida et al.,

2006). Silica degrading in electric organs may be mediated by

the CAs (silicase).

In different situations involving electric organ cryostatic

sections without treatment (from living fishes), silica was

identified along the curves of the nerves (Fig. 2b). This

occurrence raises the possibility of a fluid phase moving

through the nerves which resulted in precipitation when

suitable conditions were reached.

In contrast to the technological production and geological

synthesis of different materials (e.g. ceramics, resins, photo-

luminescent polymers, molecular sieves and catalysts) that

require extremes of temperature, pressure or pH, living systems

produce a remarkable diversity of nanostructured silicates at

ambient temperatures and pressures and at near-neutral pH

(Cha et al., 1999; Gebeshuber et al., 2003; Weaver and Morse,

2003; Schroder et al., 2007). Biogenic silica poses a challenge

for chemical synthesis or engineering (Gebeshuber et al.,

2003).

This communication provides the first evidence of

biologically produced crystalline silica mineral in cells

(electrocytes) and cholinergic nerves from living electric fish.

Acknowledgements

This research was partially supported by grants to Marıa

Prado Figueroa from the Secretarıa General de Ciencia y

Tecnica, Universidad Nacional del Sur (UNS), Bahıa Blanca,

Argentina.

Thanks are extended to Prof. E. Dominguez of the Geology

Department, UNS, who ratified our observations and allowed

for use of the microscope.

Prof. S. Fernandez-Lopez of the Geology Department,

Universidad Complutense de Madrid, is thanked for critical

review of the manuscript.

We also thank two anonymous reviewers who offered

helpful commentaries of previous versions of this manuscript.

Finally, we appreciate the CRIBABB (CONICET) for

technical assistance.

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