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Magnetic characterization of Daphnia resting eggs
BBRC Biochemical and Biophysical Research Communications 351 (2006) 566–570 www.elsevier.com/locate/ybbrc
Magnetic characterization of Daphnia resting eggs
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Masanobu Sakata a,1, Tamami Kawasaki b,f,*,1, Toshimichi Shibue c, Atsushi Takada a, Hideyuki Yoshimura e, Hideo Namiki a
f
a,d
Department of Integrative Bioscience and Biomedical Engineering, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan b Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan c Materials Characterization Central Laboratory, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan d Department of Biology, Waseda University, 1-6-1 nishiwaseda, Shinjyuku-ku, Tokyo 169-8050, Japan e Department of Physics, Meiji University, 1-1-1 Higashimita Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38, Hikari-cho, Kokubunji-shi, Tokyo 185-8540, Japan Received 8 October 2006 Available online 23 October 2006
Abstract This study characterized the magnetic materials found within Daphnia resting eggs by measuring static magnetization with a superconducting quantum interference device (SQUID) magnetometer, after forming two types of conditions, each of which consists of zerofield cooling (ZFC) and field cooling (FC). Magnetic ions, such as Fe3+, contained in Daphnia resting eggs existed as (1) paramagnetic and superparamagnetic particles, demonstrated by a magnetization and temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC, and (2) ferromagnetic particles with definite magnetic moments, the content of which was estimated to be very low, demonstrated by the Moskowitz test. Conventionally, biomagnets have been directly detected by transmission electron microscopes (TEM). As demonstrated in this study, it is possible to nondestructively detect small biomagnets by magnetization measurement, especially after two types of ZFC and FC. This nondestructive method can be applied in detecting biomagnets in complex biological organisms. 2006 Elsevier Inc. All rights reserved. Keywords: Daphnia resting egg; Magnetite; Magnetization
Under normal conditions, Daphnia reproduce by parthenogenesis. In unfavorable environments, however, Daphnia switch to sexual reproduction and produce robust resting eggs [1,2]. Each resting egg usually encases two resting embryos. When the environment becomes favorable, the eggs hatch and Daphnia reproduce cyclically by parthenogenesis again. Unique adaptive and survival abilities of Daphnia resting eggs have been previously reported. For
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Abbreviations: ZFC, zero-field cooling; FC, field cooling; SQUID, superconducting quantum interference device; TEM, transmission electron microscopes; RM, remnant magnetization; TRM, thermoremnant magnetization; FMR, ferromagnetic resonance. * Corresponding author. Fax: +81 3 5286 1507. E-mail address: [email protected] (T. Kawasaki). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.078
example, resting eggs can remain viable for decades and can withstand freezing and drying [3]. Resting eggs can also survive in the harsh environment of a predator’s digestive system [4], eventually being excreted intact. Although these unique properties of Daphnia resting eggs have been previously described, little is known about the chemical composition, microanatomy, and physical properties of the resting eggs. A previous study using a nondestructive method to measure magnetic moments with a superconducting quantum interference device (SQUID) magnetometer showed that magnetic materials, probably magnetite Fe3O4, exist in Daphnia resting eggs [5]. Biomagnets, such as magnetite, are magnetic materials that exist in bioorganisms. Biomagnets are apparently involved in behaviors such as navigation, which depends on the organism’s ability to detect or orient to the Earth’s
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
magnetic fields [6,7]. Techniques used to study biomagnets have been traditionally direct observation of micrographs and transmission electron microscopy (TEM) to detect iron by micro-analysis of magnetite. Difficulties arise when a TEM is used to observe biomagnets inside organisms more complex than bacteria. It is possible to initially overlook its existence, even though magnetic materials in fact exist within a sample. To avoid these problems, a technique that can directly and nondestructively detect magnetic materials existing in bioorganisms is needed. There are several methods that directly and nondestructively characterize magnetic materials in material science fields. In addition to simple measurement of magnetization, comparison of temperature dependence of magnetization after zero-field cooling (ZFC) and field cooling (FC) with SQUID can also detect the existence of very weak magnetic moments originating from magnetic particles in a sample and also give an indication of particle size. This method is called as the Moskowitz test [8–11]. Weiss et al. characterized ‘‘biologically controlled’’ magnetite produced by magnetotactic bacteria and ‘‘biologically induced’’ magnetite produced by dissimilatory iron-reducing bacteria, by using the Moskowitz test [9]. The Moskowitz test demonstrated that temperature dependence of magnetization differed between ‘‘biologically controlled’’ magnetite and ‘‘biologically induced’’ magnetite, which suggested that they differed in size. Thus, the Moskowitz test is effective in characterizing biomagnets. The purpose of the current study was to directly and nondestructively characterize the magnetic materials within Daphnia resting eggs by measuring static magnetization, especially after forming two types of conditions, each of which consists of ZFC and FC. In addition to the Moskowitz test, this current study examines a temperature dependence of the magnetic moments after ZFC and FC with SQUID under an applied magnetic field. These two types of magnetization enable the estimation of the total content of magnetic ions and ferromagnetic particles in Daphnia resting eggs. The current results demonstrate that the magnetic materials in Daphnia resting eggs exist mainly as paramagnetic and superparamagnetic particles. They also exist as ferromagnetic particles larger than superparamagnetic particles, of which the total content is estimated to be very low. Thus, the currently described method can be applied to nondestructively detect biomagnets in complex biological organisms. Materials and methods Daphnia magna. Daphnia magna were raised based on OECD criteria [12] with Chlorella vulgaris, maintained at 20 ± 2 C, and exposed to 16hour period of light followed by an 8-hour period of darkness per day. 50 ml of Chlorella (Chlorella vulgaris Chikugo, Chlorella Co., Tokyo) was centrifuged for 15 min at 3000 rpm. After centrifugation, the pellet was dissolved in ultrapure water and then centrifuged again for 15 min at 3000 rpm. These procedures were repeated two more times. Finally, the suspension was diluted up to 7.5 · 108 cells/ml and was used to culture Daphnia. In the current study, the Chlorella prepared by this procedure is
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Fig. 1. Variation of applied magnetic fields before and during, (a) zerofield cooling (ZFC), (b) field cooling (FC), (c) ZFC and remnant magnetization (RM) measurement at the lowest temperature, and (d) thermoremnant magnetization (TRM) measurement after FC, respectively.
called ‘‘raw Chlorella’’. To obtain resting eggs, 60 three- to four-week-old Daphnia were cultured in one liter of medium. When the population density approached the overcrowding level of 480 per liter, the Daphnia began to produce resting eggs, which were collected, rinsed with de-ionized water, and dried before measurements. Collection procedures were conducted with non-magnetic instruments. Magnetization. A SQUID magnetometer (MPMS-7, Quantum Design, CA) was used for magnetic measurement with applied field from 7 to 7 T. The samples were a set of 200 resting eggs dried in vacuum. They were set on the outer surface of a polypropylene sample tube by non-magnetic tape to minimize the effect of diamagnetism from the sample holder. In addition to magnetization measurements at 5 K, the temperature dependence of the magnetization was investigated after zero-field-cooled (ZFC) and field-cooled (FC) conditions (Fig. 1a and b). The sample was cooled from 300 K to 5 K in either zero or 7 T magnetic field, then its magnetic moment in the 7 T field was measured as a function of temperature during warming (Fig. 1a and b). In other experimental runs, the temperature dependence of either RM or TRM was recorded. To perform the Moskowitz test, the sample was first cooled down to 5 K in zero magnetic field, magnetized in the field, which was increased to 7 T, and then decreased to 0 T (<0.2 mT) to form the RM moment (Fig. 1c). In the TRM measurement, the sample was cooled to 5 K in a 7 T magnetic field, at which point the magnetic field was decreased to 0 T (<0.2 mT) (Fig. 1d). In both cases, the magnetic moments at zero applied field were measured during warming from 10 K to 300 K (Fig. 1c and d).
Results Fig. 2 shows the magnetization curve of Daphnia resting eggs obtained at 5 K. It revealed paramagnetic saturation of moment, when the applied magnetic field was stronger than about 5 T. Magnetic hysteresis was not apparent even at 5 K (Fig. 2). A previous report demonstrated that magnetization at room temperature revealed saturation of magnetic moment at about 0.2 T with no hysteresis, showing
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
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Fig. 2. Magnetization curve at 5 K of resting eggs (n = 200), (a) from 7 T to 7 T, (b) low field region of hysteresis curve. Magnetic moment unit [emu] is equal to 103 A m2.
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the existence of superparamagnetic particles in Daphnia resting eggs [5]. Therefore, it is concluded that the saturation of moment shown in Fig. 2 contains the contributions from isolated paramagnetic ions and superparamagnetic particles, which are so small that their ferromagnetic moments fluctuate randomly at 5 K. Fig. 3 shows the temperature dependence of the magnetic moments after ZFC and FC. The ZFC and FC curves were almost overlapping. This demonstrates that most of the magnetic moments after the ZFC are not frozen in random directions, but are able to rotate toward the applied
Fig. 3. Temperature dependence of magnetic moments after ZFC (d) and FC (s) (the left scale) and the reciprocals of magnetic moments after the ZFC (m) and FC (n) (the right scale). Magnetic moment unit [emu] is equal to 103 A m2.
magnetic field. Therefore, magnetic ions in the sample exist as (1) paramagnetic ions, and (2) superparamagnetic particles in the resting eggs. The reciprocals of magnetic moments at 7 T after the ZFC and FC were also plotted against absolute temperature in Fig. 3. The linear part in the range of 60–260 K was analyzed by the Curie–Weiss law v = C/(T h), where v and T are the ratio of magnetic moment to applied magnetic field (7 T = 70 kOe) and absolute temperature, respectively, assuming a linear relation between the magnetic moment and the applied field in this temperature range. The deviation from a straight line in the range of 260–300 K is not discussed here. The inverse slope C of the line is given by C ¼ Nm2eff =3k B , where N, meff, and kB are the total number of magnetic ions in the sample, the mean square of localized magnetic moments, and Boltzmann constant, respectively. The reciprocals of magnetic moments shown in Fig. 3 followed the Curie–Weiss law. These data suggest that (1) almost all the magnetic ions in Daphnia resting eggs behave paramagnetically, and (2) the content of ferromagnetic particles in the eggs is very low, if any. If the magnetic ions contained in the eggs are assumed to be Fe3+, where meff = 5lB,lB is the Bohr magneton, the total number of Fe3+ ions is estimated to be 1.3 · 1016. Fig. 4 demonstrates the temperature dependence of both remnant magnetization (RM) and thermoremnant magnetization (TRM). The ten consequential data at 0, 50, 100, and 150 K were statistically significant (t-test, p < 0.05) between RM and TRM. RM was not detected, but a small TRM was detected, suggesting that a small part of magnetic moments was fixed at the direction of applied magnetic field 7 T during FC from around 150 K. This means that magnetic particles with a definite ferromagnetic moment exist in Daphnia resting eggs. They have sizes larger than almost all the superparamagnetic particles. Based on the data from Fig. 3, total magnetic moment of the sample at 10 K was 3.2 · 104 emu. The TRM in Fig. 4 was calculated to be 1.1 · 107 emu. Therefore, the amount of magnetic ions as larger particles is about 0.03% of the total magnetic ions.
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M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
Discussion The current study characterized the magnetic materials found within the Daphnia resting eggs by static magnetization after forming two types of ZFC and FC. The Fe3+ or other magnetic ions contained in Daphnia resting eggs exist as (1) paramagnetic and superparamagnetic particles (Figs. 2 and 3), demonstrated by a magnetization and a temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC by using SQUID, and (2) ferromagnetic particles with the definite magnetic moments (Fig. 4), the content of which is very low, demonstrated by the Moskowitz test with SQUID. The magnetic moment of Daphnia resting eggs at room temperature was below detection threshold (data not shown). In contrast to the current study, a previous report showed that magnetization at room temperature revealed saturation of magnetic moment at about 0.2 T, even though a hysteresis was not detected within the limits of accuracy of the measurement [5]. There are several explanations for this discrepancy. The content of ferromagnetic particles and the total amount of magnetic ions in the current sample are much lower, compared with those of the sample in the previous study. However, the total amount of ferromagnetic particles in the previous sample is considered to have been inadequate in making a hysteresis. In the previous study, Daphnia were cultured with Chlorella tablets, whereas Daphnia in the current study were cultured with raw Chlorella. It is possible that the content and size of the magnetic particles in Daphnia resting eggs may be influenced by the difference in culture conditions. The presence of a small TRM shown in the current results (Fig. 4) suggests the existence of a small fraction of magnetic particles with magnetic moment fixed along the magnetic field during cooling at 10 K. It has been reported that magnetic materials existing in bioorganisms are mostly iron oxides, particularly magnetite (Fe3O4), in numerous species [7]. The magnetic particles existing in Daphnia resting eggs are considered to be magnetite. However, a jump of magnetization around 125 K due to Verwey transition, which is specific to magnetite, was not detected in the current study. Weiss et al. reported the result of the Moskowitz test of magnetite produced by two types of bacteria, magnetotactic and dissimilatory iron-reducing bacteria [9]. Magnetotactic bacteria produce magnetite by a ‘‘genetically controlled’’ process, possessing high crystallinity and grain size of about 50–100 nm, as shown in studies using TEM. In the case of magnetite produced by magnetotactic bacteria, the magnetization curves of RM and TRM did not overlap each other. In contrast, dissimilatory iron-reducing bacteria typically form magnetite extracellularly as a consequence of iron reduction for energy generation, a ‘‘biologically induced’’ process. ‘‘Biologically induced’’ magnetite is usually less crystalline and extremely fine-grained. In the case of magnetite produced by dissimilatory ironreducing bacteria, magnetization curves of RM and TRM
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overlapped each other. Weiss et al. also reported a Verwey transition in the temperature dependence of TRM of magnetite produced by magnetotactic bacteria [9]. Generally, Verwey transition is observed in magnetite with higher crystallinity and purity [13]. In the current study, the Moskowitz test did not show an overlapping of the magnetization curves of RM and TRM. This is similar to the Moskowitz test results of magnetotactic bacteria that Weiss et al. reported. Therefore, large magnetic particles produced by a ‘‘genetically controlled’’ process could exist in Daphnia resting eggs. If Verwey transition can be detected in Daphnia resting eggs, it will give us further insights into magnetite particle size and purity. Studies are underway to determine this. Conventionally, biomagnets have been directly detected by TEM. As demonstrated in the current study, it is possible to nondestructively detect small biomagnets by magnetization measurement. In particular, comparison of magnetizations after two types of ZFC and FC including the use of the Moskowitz test is especially effective. Magnetic resonance, particularly ferromagnetic resonance (FMR), also gives important information of magnetic materials in samples, such as magnetic particle size [9– 11,14,15]. The combination of these nondestructive methods with magnetization measurement and FMR will be more effective to identify biomagnets. Although we present on SQUID data in the current study, we are also evaluating Daphnia resting eggs using FMR. According to preliminary results, two major resonance lines near g = 2 and at g > 4.3 have been detected at 300 K. These lines are equivalent to a high-field (HF) resonance line and a low-field (LF) resonance line, which are the signature of FMR, respectively. Data for FMR including temperature dependence of the resonance lines are being analyzed and will be presented in the near future. Acknowledgments We would like to express our sincere appreciation to Kay Kohn, for guiding us through the understanding our data, discussion, and critical review of our manuscript. We also thank Seung-Yong Hahn for critical review of our manuscript and Aldric T. Hama and Erimitsu Suzuki for help with manuscript. References [1] O.T. Kleiven, P. Larsson, A. Hobæk, Sexual reproduction in Daphnia magna requires three stimuli, Oikos 65 (1992) 197–206. [2] V. Alekseev, W. Lampert, Maternal control of resting-egg production in Daphnia, Nature 414 (2001) 899–901. [3] C.E. Ca´ceres, Interspecific variation in the abundance, production, and emergence of Daphnia diapausing eggs, Ecology 79 (1998) 1699– 1710. [4] W.K. Mellors, Selective predation of ephippial Daphnia and the resistance of ephippial eggs to digestion, Ecology 56 (1975) 974–980. [5] T. Kawasaki, H. Yoshimura, T. Shibue, Y. Ikeuchi, M. Sakata, K. Igarashi, H. Takada, K. Hoshino, K. Kohn, H. Namiki, Crystalline
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[6] [7] [8]
[9]
[10]
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570 calcium phosphate and magnetic mineral content of Daphnia resting eggs, Zool. Sci. 21 (2004) 63–67. R. Blackmore, Magnetotactic bacteria, Science 190 (1975) 377. J.L. Kirschvink, Homing in on vertebrates, Nature 390 (1997) 339– 340. B.M. Moskowitz, R.B. Frankel, D.A. Bazylinski, Rock magnetic criteria for the detection of biogenic magnetite, Earth Planet. Sci. Lett. 120 (1993) 283–300. B.P. Weiss, S.S. Kim, J.L. Kirschvink, R.E. Kopp, M. Sankaran, A. Kobayashi, A. Komeili, Ferromagnetic resonance and low-temperature magnetic tests for biogenic magnetite, Earth Planet. Sci. Lett. 224 (2004) 73–89. B.P. Weiss, S.S. Kim, J.L. Kirschvink, R.E. Kopp, M. Sankaran, A. Kobayashi, A. Komeili, Magnetic tests for magnetosome chains in Martian meteorite ALH84001, Proc. Natl. Acad. Sci. USA 101 (2004) 8281–8284.
[11] H. Vali, B. Weiss, Y.L. Li, S.K. Sears, S.S. Kim, J.L. Kirschvink, C.L. Zhang, Formation of tabular single-domain magnetite induced by Geobacter metallireducens GS-15, Proc. Natl. Acad. Sci. USA 101 (2004) 16121–16126. [12] OECD Guidelines for Testing of Chemicals. Daphnia magna Reproduction Test (1998). [13] V.A.M. Brabers, F. Walz, H. Kronmu¨ller, Impurity effects upon the Verwey transition in magnetite, Phys. Rev. B 58 (1998) 14163– 14166. [14] E. Wajnberg, D. Acosta-Avalos, L.J. El-Jaick, L. Abrasad, J.L.A. Coelho, A.F. Bakuzis, P.C. Morais, D.M.S. Esquivel, Electron paramagnetic resonance study of the migratory ant Pachycondyla marginata abdomens, Biophys. J. 78 (2000) 1018–1023. [15] L.J. El-Jaick, D. Acosta-Avalos, D.M.S. Esquivel, E. Wajnberg, M.P. Linhares, Electron paramagnetic resonance study of honeybee Apis mellifera abdomens, Eur. Biophys. J. 29 (2001) 579–586.
Magnetic characterization of Daphnia resting eggs
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Masanobu Sakata a,1, Tamami Kawasaki b,f,*,1, Toshimichi Shibue c, Atsushi Takada a, Hideyuki Yoshimura e, Hideo Namiki a
f
a,d
Department of Integrative Bioscience and Biomedical Engineering, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan b Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan c Materials Characterization Central Laboratory, Waseda University, 3-4-1 Okubo, Shinjyuku-ku, Tokyo 169-8555, Japan d Department of Biology, Waseda University, 1-6-1 nishiwaseda, Shinjyuku-ku, Tokyo 169-8050, Japan e Department of Physics, Meiji University, 1-1-1 Higashimita Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38, Hikari-cho, Kokubunji-shi, Tokyo 185-8540, Japan Received 8 October 2006 Available online 23 October 2006
Abstract This study characterized the magnetic materials found within Daphnia resting eggs by measuring static magnetization with a superconducting quantum interference device (SQUID) magnetometer, after forming two types of conditions, each of which consists of zerofield cooling (ZFC) and field cooling (FC). Magnetic ions, such as Fe3+, contained in Daphnia resting eggs existed as (1) paramagnetic and superparamagnetic particles, demonstrated by a magnetization and temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC, and (2) ferromagnetic particles with definite magnetic moments, the content of which was estimated to be very low, demonstrated by the Moskowitz test. Conventionally, biomagnets have been directly detected by transmission electron microscopes (TEM). As demonstrated in this study, it is possible to nondestructively detect small biomagnets by magnetization measurement, especially after two types of ZFC and FC. This nondestructive method can be applied in detecting biomagnets in complex biological organisms. 2006 Elsevier Inc. All rights reserved. Keywords: Daphnia resting egg; Magnetite; Magnetization
Under normal conditions, Daphnia reproduce by parthenogenesis. In unfavorable environments, however, Daphnia switch to sexual reproduction and produce robust resting eggs [1,2]. Each resting egg usually encases two resting embryos. When the environment becomes favorable, the eggs hatch and Daphnia reproduce cyclically by parthenogenesis again. Unique adaptive and survival abilities of Daphnia resting eggs have been previously reported. For
q
Abbreviations: ZFC, zero-field cooling; FC, field cooling; SQUID, superconducting quantum interference device; TEM, transmission electron microscopes; RM, remnant magnetization; TRM, thermoremnant magnetization; FMR, ferromagnetic resonance. * Corresponding author. Fax: +81 3 5286 1507. E-mail address: [email protected] (T. Kawasaki). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.078
example, resting eggs can remain viable for decades and can withstand freezing and drying [3]. Resting eggs can also survive in the harsh environment of a predator’s digestive system [4], eventually being excreted intact. Although these unique properties of Daphnia resting eggs have been previously described, little is known about the chemical composition, microanatomy, and physical properties of the resting eggs. A previous study using a nondestructive method to measure magnetic moments with a superconducting quantum interference device (SQUID) magnetometer showed that magnetic materials, probably magnetite Fe3O4, exist in Daphnia resting eggs [5]. Biomagnets, such as magnetite, are magnetic materials that exist in bioorganisms. Biomagnets are apparently involved in behaviors such as navigation, which depends on the organism’s ability to detect or orient to the Earth’s
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
magnetic fields [6,7]. Techniques used to study biomagnets have been traditionally direct observation of micrographs and transmission electron microscopy (TEM) to detect iron by micro-analysis of magnetite. Difficulties arise when a TEM is used to observe biomagnets inside organisms more complex than bacteria. It is possible to initially overlook its existence, even though magnetic materials in fact exist within a sample. To avoid these problems, a technique that can directly and nondestructively detect magnetic materials existing in bioorganisms is needed. There are several methods that directly and nondestructively characterize magnetic materials in material science fields. In addition to simple measurement of magnetization, comparison of temperature dependence of magnetization after zero-field cooling (ZFC) and field cooling (FC) with SQUID can also detect the existence of very weak magnetic moments originating from magnetic particles in a sample and also give an indication of particle size. This method is called as the Moskowitz test [8–11]. Weiss et al. characterized ‘‘biologically controlled’’ magnetite produced by magnetotactic bacteria and ‘‘biologically induced’’ magnetite produced by dissimilatory iron-reducing bacteria, by using the Moskowitz test [9]. The Moskowitz test demonstrated that temperature dependence of magnetization differed between ‘‘biologically controlled’’ magnetite and ‘‘biologically induced’’ magnetite, which suggested that they differed in size. Thus, the Moskowitz test is effective in characterizing biomagnets. The purpose of the current study was to directly and nondestructively characterize the magnetic materials within Daphnia resting eggs by measuring static magnetization, especially after forming two types of conditions, each of which consists of ZFC and FC. In addition to the Moskowitz test, this current study examines a temperature dependence of the magnetic moments after ZFC and FC with SQUID under an applied magnetic field. These two types of magnetization enable the estimation of the total content of magnetic ions and ferromagnetic particles in Daphnia resting eggs. The current results demonstrate that the magnetic materials in Daphnia resting eggs exist mainly as paramagnetic and superparamagnetic particles. They also exist as ferromagnetic particles larger than superparamagnetic particles, of which the total content is estimated to be very low. Thus, the currently described method can be applied to nondestructively detect biomagnets in complex biological organisms. Materials and methods Daphnia magna. Daphnia magna were raised based on OECD criteria [12] with Chlorella vulgaris, maintained at 20 ± 2 C, and exposed to 16hour period of light followed by an 8-hour period of darkness per day. 50 ml of Chlorella (Chlorella vulgaris Chikugo, Chlorella Co., Tokyo) was centrifuged for 15 min at 3000 rpm. After centrifugation, the pellet was dissolved in ultrapure water and then centrifuged again for 15 min at 3000 rpm. These procedures were repeated two more times. Finally, the suspension was diluted up to 7.5 · 108 cells/ml and was used to culture Daphnia. In the current study, the Chlorella prepared by this procedure is
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Fig. 1. Variation of applied magnetic fields before and during, (a) zerofield cooling (ZFC), (b) field cooling (FC), (c) ZFC and remnant magnetization (RM) measurement at the lowest temperature, and (d) thermoremnant magnetization (TRM) measurement after FC, respectively.
called ‘‘raw Chlorella’’. To obtain resting eggs, 60 three- to four-week-old Daphnia were cultured in one liter of medium. When the population density approached the overcrowding level of 480 per liter, the Daphnia began to produce resting eggs, which were collected, rinsed with de-ionized water, and dried before measurements. Collection procedures were conducted with non-magnetic instruments. Magnetization. A SQUID magnetometer (MPMS-7, Quantum Design, CA) was used for magnetic measurement with applied field from 7 to 7 T. The samples were a set of 200 resting eggs dried in vacuum. They were set on the outer surface of a polypropylene sample tube by non-magnetic tape to minimize the effect of diamagnetism from the sample holder. In addition to magnetization measurements at 5 K, the temperature dependence of the magnetization was investigated after zero-field-cooled (ZFC) and field-cooled (FC) conditions (Fig. 1a and b). The sample was cooled from 300 K to 5 K in either zero or 7 T magnetic field, then its magnetic moment in the 7 T field was measured as a function of temperature during warming (Fig. 1a and b). In other experimental runs, the temperature dependence of either RM or TRM was recorded. To perform the Moskowitz test, the sample was first cooled down to 5 K in zero magnetic field, magnetized in the field, which was increased to 7 T, and then decreased to 0 T (<0.2 mT) to form the RM moment (Fig. 1c). In the TRM measurement, the sample was cooled to 5 K in a 7 T magnetic field, at which point the magnetic field was decreased to 0 T (<0.2 mT) (Fig. 1d). In both cases, the magnetic moments at zero applied field were measured during warming from 10 K to 300 K (Fig. 1c and d).
Results Fig. 2 shows the magnetization curve of Daphnia resting eggs obtained at 5 K. It revealed paramagnetic saturation of moment, when the applied magnetic field was stronger than about 5 T. Magnetic hysteresis was not apparent even at 5 K (Fig. 2). A previous report demonstrated that magnetization at room temperature revealed saturation of magnetic moment at about 0.2 T with no hysteresis, showing
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
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Fig. 2. Magnetization curve at 5 K of resting eggs (n = 200), (a) from 7 T to 7 T, (b) low field region of hysteresis curve. Magnetic moment unit [emu] is equal to 103 A m2.
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the existence of superparamagnetic particles in Daphnia resting eggs [5]. Therefore, it is concluded that the saturation of moment shown in Fig. 2 contains the contributions from isolated paramagnetic ions and superparamagnetic particles, which are so small that their ferromagnetic moments fluctuate randomly at 5 K. Fig. 3 shows the temperature dependence of the magnetic moments after ZFC and FC. The ZFC and FC curves were almost overlapping. This demonstrates that most of the magnetic moments after the ZFC are not frozen in random directions, but are able to rotate toward the applied
Fig. 3. Temperature dependence of magnetic moments after ZFC (d) and FC (s) (the left scale) and the reciprocals of magnetic moments after the ZFC (m) and FC (n) (the right scale). Magnetic moment unit [emu] is equal to 103 A m2.
magnetic field. Therefore, magnetic ions in the sample exist as (1) paramagnetic ions, and (2) superparamagnetic particles in the resting eggs. The reciprocals of magnetic moments at 7 T after the ZFC and FC were also plotted against absolute temperature in Fig. 3. The linear part in the range of 60–260 K was analyzed by the Curie–Weiss law v = C/(T h), where v and T are the ratio of magnetic moment to applied magnetic field (7 T = 70 kOe) and absolute temperature, respectively, assuming a linear relation between the magnetic moment and the applied field in this temperature range. The deviation from a straight line in the range of 260–300 K is not discussed here. The inverse slope C of the line is given by C ¼ Nm2eff =3k B , where N, meff, and kB are the total number of magnetic ions in the sample, the mean square of localized magnetic moments, and Boltzmann constant, respectively. The reciprocals of magnetic moments shown in Fig. 3 followed the Curie–Weiss law. These data suggest that (1) almost all the magnetic ions in Daphnia resting eggs behave paramagnetically, and (2) the content of ferromagnetic particles in the eggs is very low, if any. If the magnetic ions contained in the eggs are assumed to be Fe3+, where meff = 5lB,lB is the Bohr magneton, the total number of Fe3+ ions is estimated to be 1.3 · 1016. Fig. 4 demonstrates the temperature dependence of both remnant magnetization (RM) and thermoremnant magnetization (TRM). The ten consequential data at 0, 50, 100, and 150 K were statistically significant (t-test, p < 0.05) between RM and TRM. RM was not detected, but a small TRM was detected, suggesting that a small part of magnetic moments was fixed at the direction of applied magnetic field 7 T during FC from around 150 K. This means that magnetic particles with a definite ferromagnetic moment exist in Daphnia resting eggs. They have sizes larger than almost all the superparamagnetic particles. Based on the data from Fig. 3, total magnetic moment of the sample at 10 K was 3.2 · 104 emu. The TRM in Fig. 4 was calculated to be 1.1 · 107 emu. Therefore, the amount of magnetic ions as larger particles is about 0.03% of the total magnetic ions.
Magnetic moment [x 10-7 emu]
b
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Magnetic moment [x 10-4 emu]
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Fig. 4. The temperature dependence of RM after ZFC (d) and TRM after FC (s). Magnetic moment unit [emu] is equal to 103 A m2.
M. Sakata et al. / Biochemical and Biophysical Research Communications 351 (2006) 566–570
Discussion The current study characterized the magnetic materials found within the Daphnia resting eggs by static magnetization after forming two types of ZFC and FC. The Fe3+ or other magnetic ions contained in Daphnia resting eggs exist as (1) paramagnetic and superparamagnetic particles (Figs. 2 and 3), demonstrated by a magnetization and a temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC by using SQUID, and (2) ferromagnetic particles with the definite magnetic moments (Fig. 4), the content of which is very low, demonstrated by the Moskowitz test with SQUID. The magnetic moment of Daphnia resting eggs at room temperature was below detection threshold (data not shown). In contrast to the current study, a previous report showed that magnetization at room temperature revealed saturation of magnetic moment at about 0.2 T, even though a hysteresis was not detected within the limits of accuracy of the measurement [5]. There are several explanations for this discrepancy. The content of ferromagnetic particles and the total amount of magnetic ions in the current sample are much lower, compared with those of the sample in the previous study. However, the total amount of ferromagnetic particles in the previous sample is considered to have been inadequate in making a hysteresis. In the previous study, Daphnia were cultured with Chlorella tablets, whereas Daphnia in the current study were cultured with raw Chlorella. It is possible that the content and size of the magnetic particles in Daphnia resting eggs may be influenced by the difference in culture conditions. The presence of a small TRM shown in the current results (Fig. 4) suggests the existence of a small fraction of magnetic particles with magnetic moment fixed along the magnetic field during cooling at 10 K. It has been reported that magnetic materials existing in bioorganisms are mostly iron oxides, particularly magnetite (Fe3O4), in numerous species [7]. The magnetic particles existing in Daphnia resting eggs are considered to be magnetite. However, a jump of magnetization around 125 K due to Verwey transition, which is specific to magnetite, was not detected in the current study. Weiss et al. reported the result of the Moskowitz test of magnetite produced by two types of bacteria, magnetotactic and dissimilatory iron-reducing bacteria [9]. Magnetotactic bacteria produce magnetite by a ‘‘genetically controlled’’ process, possessing high crystallinity and grain size of about 50–100 nm, as shown in studies using TEM. In the case of magnetite produced by magnetotactic bacteria, the magnetization curves of RM and TRM did not overlap each other. In contrast, dissimilatory iron-reducing bacteria typically form magnetite extracellularly as a consequence of iron reduction for energy generation, a ‘‘biologically induced’’ process. ‘‘Biologically induced’’ magnetite is usually less crystalline and extremely fine-grained. In the case of magnetite produced by dissimilatory ironreducing bacteria, magnetization curves of RM and TRM
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overlapped each other. Weiss et al. also reported a Verwey transition in the temperature dependence of TRM of magnetite produced by magnetotactic bacteria [9]. Generally, Verwey transition is observed in magnetite with higher crystallinity and purity [13]. In the current study, the Moskowitz test did not show an overlapping of the magnetization curves of RM and TRM. This is similar to the Moskowitz test results of magnetotactic bacteria that Weiss et al. reported. Therefore, large magnetic particles produced by a ‘‘genetically controlled’’ process could exist in Daphnia resting eggs. If Verwey transition can be detected in Daphnia resting eggs, it will give us further insights into magnetite particle size and purity. Studies are underway to determine this. Conventionally, biomagnets have been directly detected by TEM. As demonstrated in the current study, it is possible to nondestructively detect small biomagnets by magnetization measurement. In particular, comparison of magnetizations after two types of ZFC and FC including the use of the Moskowitz test is especially effective. Magnetic resonance, particularly ferromagnetic resonance (FMR), also gives important information of magnetic materials in samples, such as magnetic particle size [9– 11,14,15]. The combination of these nondestructive methods with magnetization measurement and FMR will be more effective to identify biomagnets. Although we present on SQUID data in the current study, we are also evaluating Daphnia resting eggs using FMR. According to preliminary results, two major resonance lines near g = 2 and at g > 4.3 have been detected at 300 K. These lines are equivalent to a high-field (HF) resonance line and a low-field (LF) resonance line, which are the signature of FMR, respectively. Data for FMR including temperature dependence of the resonance lines are being analyzed and will be presented in the near future. Acknowledgments We would like to express our sincere appreciation to Kay Kohn, for guiding us through the understanding our data, discussion, and critical review of our manuscript. We also thank Seung-Yong Hahn for critical review of our manuscript and Aldric T. Hama and Erimitsu Suzuki for help with manuscript. References [1] O.T. Kleiven, P. Larsson, A. Hobæk, Sexual reproduction in Daphnia magna requires three stimuli, Oikos 65 (1992) 197–206. [2] V. Alekseev, W. Lampert, Maternal control of resting-egg production in Daphnia, Nature 414 (2001) 899–901. [3] C.E. Ca´ceres, Interspecific variation in the abundance, production, and emergence of Daphnia diapausing eggs, Ecology 79 (1998) 1699– 1710. [4] W.K. Mellors, Selective predation of ephippial Daphnia and the resistance of ephippial eggs to digestion, Ecology 56 (1975) 974–980. [5] T. Kawasaki, H. Yoshimura, T. Shibue, Y. Ikeuchi, M. Sakata, K. Igarashi, H. Takada, K. Hoshino, K. Kohn, H. Namiki, Crystalline
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