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Non-destructive method to study the internal anatomy of the Chilean scallop Argopecten purpuratus.

ABSTRACT Scallop aquaculture has a 20-y-old history in Coquimbo, Chile. At the beginning develop and introduce on industrial level the culture techniques was the main goal, but in recent years research to improve the broodstock quality has been introduced. Development of non destructive procedures to determine gonadal ripeness were necessary. The use of magnetic resonance imaging proved to be an interesting tool to view the internal anatomy of the chilean scallop Argopecten purpuratus (Lamarck 1819) without any harmful side effect. A central groove located in the middle of the adductor muscle, became visible using MRI, and could possibly be related to the haemolymphatic circulation system.

KEY WORDS: scallops, non-invasive analysis, magnetic resonance imaging (MRI)

INTRODUCTION

Evaluation methods to determine adductor muscle and gonad size in the Northern Chilean scallop are generally destructive, but the information is needed for production and research purposes. Classically, the investigation of soft tissue in marine molluscs and especially in marine bivalves relies on destructive methods, because a shell completely encloses the animal. Anatomical structures are generally studied after removing the shell, followed by dissection by mean of histologic serial sections generally with a resolution of 4-5 [micro]m (Chavez-Villalba et al. 2002). One of the difficulties presented by scallops is that to check ripeness of the gonads, the shells have to be separated, and the animal dies. On the other hand, the valve opening, as seen in Figure 1 is not enough to assess with certainty the adequate ripeness of the gonad to induce spawning with good results, having a better view (Fig. 2) if valves are completely separated.

Animals produced through several biotechnological processes, or those potential high quality specimens obtained through genetic breeding programs should not be killed unless totally necessary. Searching available harmless methods of analysis, we tried Magnetic Resonance Imaging (MRI), a well-established, noninvasive imaging modality for the central nervous system in humans (Tyszka et al. 2005). Noninvasive imaging technologies such as MRI, Magnetic Resonance Microscopy (MRM) and Nuclear Magnetic Resonance (NMR), are all increasingly in demand by researchers in many biological disciplines (Robinson et al. 2000, Barkovich, 2002, Almeida de Oliveira et al. 2006, Hakum/iki & Brindle 2003, Tyszka et al. 2005, Holliman et al. 2008).

MRI is a non-destructive procedure, and in marine sciences, MRI has been used to detect the effect of marine toxins with the aid of different isotopes (Agafonova et al. 2008). This technique has been used to study the internal anatomy of oysters (Pouvreau et al. 2006) and recently in freshwater mussels (Holliman et al. 2008), but this is the first analysis in scallops.

MRI is based on the principles of nuclear magnetic resonance (NMR), a spectroscopic technique used by scientists to obtain microscopic chemical and physical information about molecules. MRI creates images using the principles of Nuclear Magnetic Resonance, and it is possible in the scallop, because its soft-tissued body is filled with small biological "magnets," the most abundant and responsive of which is the proton, the nucleus of the hydrogen atom, and it possesses a nonzero angular momentum or nuclear spin, which creates a magnetic dipole moment along their rotational axis. When it is placed in an external magnetic field, a net nuclear magnetization is created by the equilibrium difference between the magnetic dipole moments of spin populations in two different energy states. This net magnetization can be perturbed by electromagnetic radiation like radio frequencies. Perturbed magnetization and its recovery (relaxation) to the equilibrium state can be detected and information on the nuclear spin environment of the sample or tissue can be obtained. This process is the Nuclear Magnetic Resonance, and its signal can be used to detect and quantify the presence of distinct chemical compounds, the so called Nuclear Magnetic Resonance Spectroscopy or MRS for short. The other possibility is to create images based on the spatial distribution and/or relaxation of the observed nuclei, the MRI (Hakumaki & Brindle 2003). Once the organism is placed in the cylindrical magnet, the process follows three basic steps: (1) MRI creates a steady state within the organism by placing it in a steady magnetic field that is 30,000 times stronger than the earth's magnetic field. (2) MRI stimulates the organism with radio waves to change the steady-state orientation of protons. (3) It then stops the radio waves and "listens" to the body's electromagnetic transmissions at a selected frequency. The rate at which protons return to their ground-stage of relaxation after perturbation by a radio wave is influenced by solution composition and concentration, pH, viscosity, and cell structure. The transmitted signal is used to construct internal images.

[FIGURE 1 OMITTED]

These images are topological representations of mobile water fractions in soft-tissue specimens. Image intensities at discrete locations throughout the sample are sensitive to both instrument settings and sample-dependent such as proton spin density or water content.

MATERIAL AND METHODS

Juvenile (3-cm shell height) and adult scallop (7-8-cm shell height) scallops (Argopecten purpuratus, Lamarck 1819) were transported alive from Coquimbo to Santiago in a styrofoam box with layers of foam wetted with microfiltered sea water. The transport was done during the night to avoid unnecessary exposure to heat.

After arrival at the MRI center (14 h after collection) they were placed on a plastic sponge in a 2-L plastic container, filled with microfiltered seawater. The container was closed with a lid and placed inside the tunnel of the Magnetic Resonator (Philips T5--Intera) (Fig. 3), using a cylinder for knee examination (Fig. 4). The magnetic pulses are programmed and registered at the Pulse Programming workstation. After receiving the magnetic signal, the image is processed and can be analyzed using image analysis software, to construct the final picture.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

After the examination was completed, the scallops were fed with a concentrated suspension of live microalgae to determine, through there feeding behavior, how well they endured the procedure.

RESULTS

Multiple thin sections from different angles and directions were obtained, being the images from adult scallops better in resolution then the juveniles. The smaller scallops were very active during examination time, producing a blurred image. The adult scallops lay still, and good quality images of the different slices could be recorded.

[FIGURE 4 OMITTED]

The Figure 5, shows six MRI images from adult scallops that showed recognizable shell (S), kidney (K), adductor muscle (AM), crystalline style (CS), male and female gonad portion (MF), digestive gland (DG) and a groove (Gr) located in the central area of the adductor muscle.

DISCUSSION

The results obtained in this preliminary work show that MRI can be easily obtained for scallops, using equipment for medical purposes without any additional requirement. Each organ was clearly recognizable, and the observation of a groove in the central area of the adductor muscle, indicates the possible presence of a vein type structure that could be part of the hemolymphatic system. This groove could not be seen in the macroscopic view of the whole adductor muscle (Fig. lb). It is generally accepted that scallops have an open circulatory system, but there are some structures.

Better images were obtained with larger animals, probably because they were immobile. Small or juvenile scallops are very active and tend to swim around, causing the blurred images. To be able to achieve good quality images, these samples should be anaesthetized or examined only on wet foam, instead of using a container filled with seawater, to avoid swimming. The scallops (adults and juveniles) were not affected by the analysis, shown by the active feeding behavior after the MRI procedure was completed.

MRI is an unexplored procedure to study live marine organisms, and it could become an interesting tool, when images are correlated with other biological information such as biochemical composition, gonad development, diseases, nutritional status, and contamination. These correlations can be used to establish a non-destructive and a non-invasive technique to assess, for example, adequate gonadal condition to induce spawning at precise moments, without stressing and risking valuable broodstock unnecessarily. On the other hand, the size of adductor muscle could be correlated to its weight, another important feature for selection purposes.

ACKNOWLEDGMENT

The authors thank Dr. S. Shumway for her comments and encouragement. These first analysis were possible through the collaboration of two projects funded by FONDEF (Chile) D02 1 1095 and D9911018.

[FIGURE 5 OMITTED]

LITERATURE CITED

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Almeida de Oliveira, S. L. H. W. Gowdak, G. Buckberg, J. E. Krieger & The RESTORE Group. 2006. Cell biology, MRI and geometry: insight into a microscopic / macroscopic marriage. Eur. J. Cardiothorac. Surg. 295:5259-5265.

Barkovich, A. J. 2002. Magnetic resonance imaging: role and understanding of cerebral malformations. Review Article. Brain Dev. 24: 2-12.

Chavez-Villalba, J., J. Pommier, J. Andriamiseza, S. Pouvreau, J. Barret, J. C. Cochard & M. LcPennec. 2002. Broodstock conditioning of the oyster Crassostrea gigas: origin and temperature effect. Aquaculture 214:115-130.

Hakumaki, J. M. & K. M. Brindle. 2003. Techniques: Visualizing apoptosis using nuclear magnetic resonance. Trends Pharmacol. Sci. 24:146-149.

Holliman, F. M., D. Davis, A. E. Bogan, T. J. Kwak, W. G. Cope & J. F. Levine. 2008. Magnetic Resonance imaging of live freshwater mussels (Unionidae). Invertebr. Biol. 127:396-402.

Pouvreau, S., M. Rambeau, J. C. Cochard & R. Robert. 2006. Investigation of marine bivalve morphology by in vivo MR imaging: First anatomical results of a promising technique. Aquaculture 259:415-423.

Robinson, A., C. J. Clark & J. Clemens. 2000. Using 1 H magnetic resonance imaging and complementary analytical techniques to characterize developmental changes in the Zantedeschia Spreng. Tuber. J. Exp. Bot. 51:2009-2020.

Tyszka, J. M., S. E. Fraser & R. E. Jacobs. 2005. Magnetic Resonance microscopy: recent advances and applications. Curr. Opin. Biotechnol. 16:93-99.

ELISABETH VON BRAND, (1) * MAGDALENA CISTERNA, (1, [dagger]) GERMAN MERINO, (2) EDUARDO URIBE, (2) CLAUDIO PALMA-ROJAS, (3) MATIAS ROSENBLITT (4) AND JOSE LUIS ALBORNOZ (4)

(1) Departamento de Biologia Marina, Facultad de Ciencias del Mar, Universidad Catolica del Notre, Casilla: 117 Coquimbo, Chile; (2) Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Catolica del Norte, Casilla: 117 Coquimbo, Chile; (3) Departamento de Biologia, Facultad de Ciencias, Universidad de La Serena, Casilla: 588 La Serena, Chile; (4) Pontificia Universidad Catolica de Chile, Centro de Investigacion en Resonancia Magnetica, Vicuna Mackenna 4686, Santiago, Chile

* Corresponding author. E-mail: evonbran@ucn.cl

([dagger]) Present address: Facultad de Educacion, Universidad Alberto Hurtado, Erasmo Escala 1825, Santiago, Chile.
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Article Details
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Author:von Brand, Elisabeth; Cisterna, Magdalena; Merino, German; Uribe, Eduardo; Palma-rojas, Claudio; Ros
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:3CHIL
Date:Apr 1, 2009
Words:1745
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