**2.1** *Psammobatis extenta***: A Rajidae family fish**

Adult female and male *P. extenta* were collected from the Bahía Blanca Estuary (38° 40'S and 39°30'S, 62°16'W and 63°26'W) in the Buenos Aires Province, 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°C.

Electrocytes from *P. extenta* are cup-shaped cells, multinucleated and polarized. They have an anterior, concave, innervated face and a posterior, convex, non-innervated face, that shows a very large system of caveolae (Prado Figueroa *et al*., 1995). These cup-shaped electrocytes are plesiomorphic, phylogenies based on morphological data (Jacob *et al*., 1994). Neuronal cell death and synaptic terminal degeneration have been noted in the adult electric organs of fish from the Rajidae family (Fox *et al*., 1990 and our observations). Understanding cellular and molecular mechanisms participating in neurodegenerative process is thus an important field of research.

#### **2.2 Silicon in electric organs**

286 Advances in Crystallization Processes

shape, 12–15 micron in size, translucent, with a low refraction index. The crossed-polarizer

In this chapter, we document the visualization and identification of chalcedony crystals in electric organs, by using a Leica TCS - SP2 Laser Scanning Confocal Microscope (LSCM). Three ion lasers were used i.e.: argon with emission band in 458 nm (cyan), 476 nm (bluegreen), 488 nm (green) and 514 nm (yellow); He/Ne in 543 nm with emission band in red, and He/Ne with emission band in 633 nm (blue). The autofluorescent character of chalcedony (a mineral) allowed us to obtain images of the crystals together with a topographic study. Chalcedony consists of nanoscale intergrowths of silica polymorphs: quartz and moganite (Heaney & Post, 1992). These silica polymorphs, with their two different nanocrystal structures, are described in the present chapter. Quartz and moganite are both identified in three-dimensional (3-D) images using a LSCM and Leica software. 3-D images were generated as "surface": the blank spaces between the pixels are filled. 3-D images were also generated as "wireframe": all pixels are linked with lines, while the blank spaces remain free. Many images generated by differential interference contrast (DIC) are

Biomineralization by silica is a complicated process observed in living organisms. Of the intriguing topics that are receiving renewed attention, the study of biomineral formation based on organic templates is one of the most fascinating topics today (Ehrlich, 2010; Ehrlich *et al*., 2010). Biosilicification is an evolutionarily old and widespread type of biomineralization both in unicellular and multicellular organisms, including sponges,

We have studied *Psammobatis extenta* electric organs from the Rajidae family, a group of elasmobranch electric fish. Electric organs are structures specialized in the production of electric discharges (Fessard, 1958). Their major cell components, called electrocytes, are highly polarized. We could detect biomineralization by microcrystalline silica in *P. extenta*

Adult female and male *P. extenta* were collected from the Bahía Blanca Estuary (38° 40'S and 39°30'S, 62°16'W and 63°26'W) in the Buenos Aires Province, Argentina and transported to

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

Electrocytes from *P. extenta* are cup-shaped cells, multinucleated and polarized. They have an anterior, concave, innervated face and a posterior, convex, non-innervated face, that shows a very large system of caveolae (Prado Figueroa *et al*., 1995). These cup-shaped electrocytes are plesiomorphic, phylogenies based on morphological data (Jacob *et al*., 1994). Neuronal cell death and synaptic terminal degeneration have been noted in the adult electric organs of fish from the Rajidae family (Fox *et al*., 1990 and our observations).

the laboratory in sealed polyethylene bags containing oxygen-saturated seawater.

diatoms, radiolarians, choanoflagellates, and higher plants (Schroeder *et al*., 2008).

image shows first order birefringence colour (grey–white) and radial extinction.

also shown in this chapter.

**2. Biomineralization by silica in electric fish** 

electric organs (Prado Figueroa *et al*., 2008).

nitrogen at -198°C.

**2.1** *Psammobatis extenta***: A Rajidae family fish** 

Silicon is an essential element for animals (Carlisle, 1982). Silicon may function as a biological cross-linking agent and may contribute to the architecture and resilience of connective tissue (Schwarz, 1973). It has also been documented that silicon is present as a silanolate, i.e., an ester-like derivative of silicic acid and plays a role in the structural organization of glycosaminoglycans and polyuronides (Schwarz, 1973).

We could observe aluminium and silicon in the cytoplasmic extracts of *P. extenta* electric tissue (Prado Figueroa *et al*., 2008) using a combination of scanning electron microscopy and X-ray spectrometry (EDS/SEM) (see Fig. 1). The result of this microanalysis is an energydispersive spectrum in which the peaks are localized at energy lines characteristic for each element

Fig. 1. Electric organ cytoplasmic extracts from *Psammobatis extenta* on lyophilised paper and metalized with gold were microanalyzed by using EDS/SEM. This energy dispersive spectrum (a) shows high peaks localized at energy lines characteristic for oxygen, silicon and aluminium. Sodium, potassium, calcium and zinc are also observed. Weight and atomic percents for these elements are indicated next to the spectrum (b).

The Growth of Chalcedony (Nanocrystalline Silica) in Electric Organs from Living Marine Fish 289

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 Leica polarized light microscope (DMLP). This microscope 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 360º rotation, minerals in different positions display their optical properties, such as birefringence colour and extinction position, with crossed polarizers.

All electric organ fractions in crossed-polarizers show SiO2 replacements in grey and white arrangement, with undulatory extinction. The crossed-polarizer image of a cytoplasmic extract shows SiO2 replacements in grey and white arrangements (chalcedony), Fig. 2.

Electric organs without any treatment were also used for X-ray diffraction analysis. Different peaks were obtained from diffractometric analysis; specifically peaks belonged to a

Autofluorescence characteristics of minerals have been described by Henkel (1989). In this chapter, we document the visualization and identification of chalcedony crystals in electric organs, by using a Leica TCS - SP2 Laser Scanning Confocal Microscope (LSCM). This microscope has three ion lasers i.e.: argon with emission band in 458 nm (cyan), 476 nm (blue-green), 488 nm (green) and 514 nm (yellow); He/Ne with emission band in 543 nm (red) and He/Ne with emission band in 633 nm (blue). Since chalcedony is characteristically

Adult female and male *P. extenta* were collected from Bahía Blanca Estuary in Buenos Aires Province, Argentina. Fractionation of electric tissue homogenates by differential

Fractions (nuclear fraction "N"; microsomes "P" and supernatant "S") were used and observed with a LSCM. A nuclear fraction shows many autofluorescent crystals and also little electrocytes, Fig. 3. Electrocytes from the electric organ of the Patagonian ray *Psammobatis extenta* are very unusual cells: semicircular in shape, multinucleated and highly

Fig. 3 shows an unbroken electrocyte with many autofluorescent crystals, these were observed with an argon ion laser with emission band at 488 nm (green). This image was

Normally, the microsomal fraction contains membranes from the synaptic region. The microsomal fraction has many autofluorescent crystals. A crystal of chalcedony from the microsomal fraction, its dimensions and autofluorescent intensity (I, arbitrary units) are

polarized. Their anterior face is concave and innervated by numerous nerve-endings.

quartz (low quartz; Moore and Reynolds, 1997). (See: Prado Figueroa *et al*., 2008).

**3. Autofluorescent crystalline silica detected by using a LSCM** 

an autofluorescent mineral, we were allowed to obtain images of crystals.

**3.1 Autofluorescent microcrystalline silica (chalcedony)** 

centrifugation was carried out as described in Section 2.1.

merged with (DIC).

An electric organ cytoplasmic extract (50 μl) on lyophilised paper (Labconco Corp., USA) was fixed in 2.5% glutaraldehyde in a 0.05 M sodium phosphate buffer (pH 7.2) for 60 min at 4 °C. Samples were washed with buffer and bi-distilled water for 2 h, then, dehydrated. (For more detail of this method, see Prado Figueroa *et al*., 2008).

This method (EDS/SEM) has also been used for studying photosensitizers in electric tissue (Prado Figueroa & Santiago, 2004).
