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Upside-down gliding of Lymnaea.

Introduction

The pond snail Lymnaea stagnalis is most often thought of as a model system by which to study the causal mecha nisms that underlie learning and memory (Azami et al, 2006; Kemenes et al, 2006; Sugai et al, 2007; Orr and Lukowiak, 2008). Lymnaea, however, is capable of per forming many other interesting behaviors, such as gliding upside down just beneath the water surface. This form of locomotion can be observed both in the field and in aquaria in the laboratory (Fig. 1). We have termed this behavior "upside-down gliding" or "back-swimming." Often snails appear to be floating passively, particularly if there is a current; but at other times active locomotion appears to occur. Little is known about the mechanism of this active upside-down gliding. In addition, other befraviors such as aerial respiration and copulation also come about when snails are in this upside-down posture clinging to the underside of the water surface. We therefore initiated a series of studies to help elucidate the means by which snails are able to locomote upside down at the undersurface of the water.

[FIGURE 1 OMITTED]

In laboratory aquaria, Lymnaea typically locomotes along the sides of the aquarium, on top of the bottom substrata, and on food such as lettuce floating on the surface of the water. This locomotory behavior is thought to be the result of snails gliding over the mucus trail secreted from the foot onto the substrate they are crawling on. This gliding is accomplished by both ciliary activity on the foot and peri staltic contraction of the foot muscles (Miller, 1974). A cluster of serotonergic neurons in each of the left and right pedal ganglia innervate the foot and control the beating of its cilia (Syed et al., 1988; Syed and Winlow, 1989). A more recent report has indicated that an octopaminergic neural network might also be involved in controlling locomotory activity (Ormshaw and Elliott, 2006). In addition, in the embryo of pond snails, ciliadriven rotation behavior is regulated by either serotonin or doparaine (Uhler et al., 2000; Doran et al., 2004). Taken together, these results indicate that specific neurons using different biogenic amines are able to regulate cilia activity and thus control locomotory behavior.

Previous observations of upside-down gliding have indicated that mucus is also secreted from the foot during this motion. We hypothesized that this mucus adheres to the underside of the water surface and allows snails to locomote at it upside down. We therefore needed both to characterize this mucus and to observe the structure and movement of the foot of Lymnaea on this mucus. The data presented here show that beating cilia provide the main driving force for upside-down gliding and that peristaltic contraction of the foot muscles plays a diminished role in this behavior com pared to its prominent role in normal locomotion. Thus, these data are consistent with the hypothesis that Lymnaea secretes mucus from its foot and that this mucus sticks to the undersurface of the water, enabling snails to perform up-side-down gliding propelled by ciliary beating.

Materials and Methods

Speed of upside-down gliding

Specimens of Lymnaea stagnalis L. with shell lengths of 15-25 mm were obtained from our snail-rearing facility (original stocks from Vrije Universiteit Amsterdam). All snails were maintained in dechlorinated tapwater (i.e., pond water) and fed lettuce. These snails were maintained in a container containing dechlorinated tapwater at 16-22 [degrees]C to observe upside-down gliding. The experiments were per-formed at three times of day: 1000-1300, 1300-1600, and 1600-1900 h. The container was covered with a transparent acrylic plate and transparent food-wrap. We used a felt-tip pen to trace the movements of the snails on the food-wrap while timing the duration of the locomotory movement. in this way we were able to calculate the speed of the upside down gliding.

Effects of a detergent on upside-down gliding

When a snail started gliding upside down in the container, a 100-[micro]l drop of detergent, polyxyethylene (20) sorbitan monolaurate (Tween 20; concentration 0.01% to 100%; Wako, Osaka, Japan), was applied to the foot of the snail. The experiments were performed at a water temperature of 22-28 [degrees]C during the day. Because a detergent is referred to as a surface-active agent, as the control solutions we used a surface-active substance (ethanol), a surface-inaetive sub stance (NaCl), and distilled water (DW).

Speed of locomotion on hydrophobic or hydrophilic substrates

We measured the walking speed of Lymnaea on both hydrophobic and hydrophilic plates. The hydrophobic plate was polytetrafluorethylene (Teflon), and the hydrophilic plate was silicone. These experiments were carried out in air. That is, snails were taken from their home aquaria and placed directly onto the specific plate. Thus, the only water present was that which was on the snail when it was transferred to the plate. These experiments were performed at a room temperature of 17-22 [degrees] C during the daylight hours.

Scanning electron microscopy

To observe the surface morphology and fine structures of the foot of Lymnaea, we employed scanning electron microscopy. The specimens were prepared as follows. The whole body of the snail was fixed in half-strength Kar-novsky's fixative (2.0% paraformaldehyde and 2.5% glutaraldehyde), and the foot excised from its body was postfixed in 10% glutaraldehyde. The postfixed specimens were dehydrated in an ethanol series (50%, 70%, 90%, and three changes of 100%) for 20 min each, then infiltrated with t-butyl alcohol. Although it is difficult to absolutely separate mucus from specimens, we are confident that fixation utilizing a series of ethanol and t-butyl alcohol removed the majority of the mucus, which consequently did not affect the obtained images. After they were freeze-dried, the specimens were mounted onto a sample holder and coated with platinum (about 12 nm). The prepared specimens were observed with a scanning electron microscope (JSM T330; JEOL, Tokyo, Japan). All images were taken at an angle of 45 degrees.

Interference reflection microscopy

Interference reflection microscopy (1RM) has been used to visualize cell-substrate adhesion {i.e., focal contacts) in living cells in culture (Curtis. 1964; Abercrombie and Dunn, 1975). We applied IRJV1 to visualize the morphology of the interface between a snail's foot and a glass substrate. Because the adherence of snails to a thin glass plate is required for observation by IRM, a few snails were placed into a coverslip-bottom dish containing dechlorinated tapwater. When the snails adhered to and locomoted on the coverslip, spatial and temporal changes in the morphology of the snail's foot on the coverslip surface were observed by IRM. The apparatus used here was composed of an inverted optical microscope (IX81; Olympus, Tokyo, Japan), a 100X oil immersion objective lens (Olympus), and the optics for interference reflection microscopy (Olympus). The images were taken with a digital CCD camera (DP70; Olympus). To assess the temporal changes in morphology, we collected time-lapse images at intervals of 1/15 s for 10 s. The sequential images were converted into a real-time movie at 15 frames per second with MediaStudio Pro 7 software (Ulead Systems, Yokohama, Japan).

Statistical analyses

Data were expressed as the mean [+ or -] SEM. The statistical significance was evaluated by Student's t-test or ANOVA and post hoc Scheffe test or Fisher's exact probability test. Significance was at the 0.05 level.

Results

Speed of upside-down gliding

The speed of upside-down gliding of Lymnaea was calculated at three times of the day: 1000-1300, 1300-1600, and 1600-1900; and at three temperatures: 16-18 [degrees] C, 18-20 [degrees] C, and 20-22 [degrees] C (Fig. 2). Our overall impression was that upside-down gliding was faster in the morning (e.g., 1.53 [+ or -] 0.22 mm [s.sup.-1] for 1000-1300 at 16-18 [degrees] C) than in the evening (e.g., 0.79 [+ or -] 0.08 mm [s.sup.-1] for 1600-1900 at 18-20 [degrees] C). Despite this general impression, however, the only statistically significant difference in the speeds was between the time ranges of 1000-1300 (speed of 1.18 [+ or -] 0.11 mm [s.sup.-1]) and 1600-1900 (0.79 [+ or -] 0.08 mm [s.sup.-1]) at a temperature of 18-20 [degrees] C (P < 0,01 by ANOVA and post hoc Scheffe test). On the other hand, water temperature did not seem to have any clear effect on the gliding speeds.

[FIGURE 2 OMITTED]

Effects of a detergent on upside-down gliding

When individuals of Lymnaea glide upside down at the water surface or locomote on an aquarium wall, they appear to secrete mucus from the foot. We thus attempted to confirm the secretion of mucus from the foot, and to determine whether this mucus plays an important role in allowing the snail to utilize the surface tension of the water. We succeeded in isolating the mucus from the foot with a pipette.

To characterize the mucus secreted from the foot, we applied a single drop (100 [micro]l) of Tween 20 onto the foot of an upside-down-gliding snail and observed the effects. Because surface tension causes the surface of the water to behave like an elastic sheet, disruption of it (e.g., use of a detergent) should cause any object clinging to it to be repelled from the site of disruption. If the mucus assists in the maintenance of surface tension, the application of detergent would cause the snail to be repelled while maintaining its upside-down gliding, because the detergent reduces the surface tension of water and thus would cause the snail to drift away from the point of contact with the detergent.

We used five concentrations of Tween 20: 100%, 10%, 1%, 0.1%, and 0.01%. Five experiments were performed at each concentration. At the concentrations of 100%, 10%, 1 %, or 0.1 %, 18 of 20 snails were repelled from the point of application (Fig. 3), whereas 0.01% Tween 20 did not induce any alteration in the upside-down gliding (P < 0.01 by Fisher's exact probability test). Interestingly, even 100% Tween 20 did not sink the snails gliding upside down.

[FIGURE 3 OMITTED]

Because a detergent is a surface-active agent (i.e., surfactant), we also applied a surface-active substance (etha nol), a surface-inactive substance (NaCl), and distilled water (DW) as controls. The concentrations of ethanol applied were 100% in = 5 experiments), 10% (n = 5), and 1% (n = 5); and those of NaCl solutions were 50 [micro]xmol [l.sup.-1] (n = 7) and 5 [micro]mol [l.sup.-1] (n = 6). The 100% ethanol appeared toxic for Lymnaea because some snails died after its application, and so the data for this concentration were not entered into the analysis. The application of 10% ethanol repelled the snails but also caused 3 of the 5 snails tested to gyrate at the water surface, whereas the 1% solution evoked no reaction in the 5 snails (P < 0.05 by Fisher's exact probability test).

The application of a 50 [micro]mol [1.sup.-1] NaCl solution and of a 5 [micro] mol [1.sup.-1] solution initially stopped the upside-down gliding, but snails quickly resumed their gliding (6 out of 7 snails for the 50 [micro] mol [1.sup.-1] solution, and 1 out of 6 for the 5 [micro]mol [1.sup.-1] solution). However, no snail was repelled by the application of a NaCl solution (0 of 13 snails). DW application also did not repel Lymnaea (0 of 4 snails).

We interpreted the above data as meaning that the mucus secreted from the foot during upside-down gliding indeed assists in the use of the surface tension of the water. That is, the mucus repels the water; in other words, the wettability of mucus is low.

Locomotory speed on hydrophobic or hydrophilic plates

We next examined the effects of this mucus on normal locomotion (e.g., along the substratum or along the sides of an aquarium wall). For this analysis, we chose two materials: polytetrafluorethylene (Teflon) and silicone. Teflon is hydrophobic and silicone is hydrophilic.

We calculated the speed of snails on the Teflon plates to be 0.47 [+ or -] 0.05 mm [s.sup.-1](n = 17), whereas the speed on the silicone plates was 0.50 [+ or -] 0.05 mm [s.sup.-1] (n = 19). These values are not significantly different from each other (Student's t-test).

The foot cilia of Lymnaea

In normal locomotion (or right-side-up gliding), both ciliary beating and peristaltic movement of the foot muscles provide the driving force for locomotion. In contrast, in upside-down gliding, the beating cilia appear to act as the main driving force with only a small contribution from peristaltic contraction. We thus examined the foot of Lym-naea by scanning electron microscopy.

A typical low-magnification image is shown in Figure 4A. The bright area, indicated by the arrowheads, at the left of the image corresponds to the periphery of the foot. An enlargement of this image reveals that the foot is not a smooth surface, but rather is covered with numerous cilia (Fig. 4B). The cilia are distributed unevenly over the surface of the foot, with some areas showing dense packing of cilia (10 [micro]m). In these areas, cilia appear to be grouped into parallel or twisted-together bundles of about 10 cilia. The diameter of individual cilia was on the order of a few hundreds of nanometers, whereas the length was more than 10 [micro]m (Fig. 4C).

[FIGURE 4 OMITTED]

Movement of cilia

Interference reflection microscopy offered us a chance to observe the movement of the foot cilia. At the boundary between the foot and the glass substrate, a great number of high-contrast filamentous structures (< 1 [micro],m in diameter) were observed (Fig. 5A). These high-contrast filamentous structures are the result of cilia attaching to the glass substrate. We found that some cilia were attached to the glass while others were not. The cilia attached to the glass appeared to form island-like regions. The size of these is landlike structures is in good agreement with the 10-[micro]m-order irregularities seen in the scanning electron micrographs (Fig. 4B). The data indicate that the whole foot does not adhere to the substrate in the manner of a sucker, but that the foot adheres to the substrate via punctate adhesion structures. We observed peristaltic muscle movement on the slide glass when the snail was performing normal locomotion and found that the foot was out of focus when there was a muscle contraction.

[FIGURE 5 OMITTED]

In Figure 5, we show a typical series of foot movements as observed by interference reflection microscopy; during this series, the snail moved from the right to the left side and then stopped (see also Movie, http://www.biolbull.org/ supplemental/). The sampling time for the series was 5 s. As can be seen, most of the cilia were oriented parallel to the direction of movement, but some of the cilia in island-like regions were oriented differently (see the ovals in Fig. 5B, C). During this particular movement, the cilia oriented parallel to the movement swept over the glass substrate, while those situated in island-like regions beat in a periodic and coordinated manner reminiscent of paddling. When the snail stopped moving, both populations of cilia (gliding and beating) also stopped. As mentioned earlier, the speed of locomotion was about 0.5 mm [s.sup.-1] . However, because this speed was faster than we can detect with interference reflection microscopy, we captured the images shown in Figure 5 and Movie (http://www.biolbull.org/supplementai/) when the snail was moving at a slower speed.

Discussion

In the present study, we examined a snail behavior that has not been looked at to any great extent before--namely, upside-down gliding. Lymnaea makes use of this behavior to move upside down by clinging to the underside of the water surface. This ability, much like a water stridor's ability to "walk on water," makes use of the surface tension of water. In order for Lymnaea to move along the underside of the water surface, it must secrete mucus that attaches to the surface and allows the cilia on the foot to propel the snail along. Here we measured the speed at which Lymnaea can move by upside-down gliding, and we also partly characterized the mucus secreted from the foot that enables the snail to perform this behavior.

We found that the speed at which the snails can locomote using upside-down gliding was dependent on both the time of day and the water temperature. Snails tested at a water temperature of 18-20 [degrees]C moved significantly faster in the morning than just before dark (Fig. 2). These data on speed of locomotion and time of day are consistent with data obtained earlier (Wagatsuma et al., 2004). These findings showed that Lymnaea has a circadian rhythm of activity, with the peak of activity occurring in the morning, and also that these snails learn and remember more effectively if trained in the morning than if trained toward dusk. It is of interest, however, that in water maintained at a temperature greater than 20 [degrees]C, this relationship {i.e., faster in the morning) was not seen. It could be that, in snails chronically maintained at 16-18 [degrees]C, the circadian clock is upset by an acute test at a temperature over 20 [degrees]C. This issue will have to be investigated in the future.

Like other molluscs, Lymnaea possesses extraocular photoreceptors (Stoll, 1973; Orr et al., 2007). We have begun to electrophysiologically examine how the extraocular dermal photo-input is conveyed to the central nervous system (Chono et al., 2002). Photic stimulation of the foot evokes inhibitory inputs to neurons in the pedal ganglia via the inferior pedal nerves. This inhibitory activity is specific to the dermal photoreceptors in the foot and is not due to photo stimulation of the eyes. Because pedal ganglion neurons play a large role in the mediation of locomotory activity in the snail, it is plausible that Lymnaea senses light by the extraocular dermal photoreceptors in the foot during upside-down gliding. We hypothesize that this ability to detect changes in illumination by dermal photoreceptors located in the foot may play a role in behaviors associated with predator detection (Orr et at, 2007).

The second question we investigated was how the snails adhere to the undersurface of the water and then glide upside-down. We found that we could isolate mucus that was secreted from the foot of the snail. However, we did not attempt to discover the composition of this mucus, other than to determine that it has little wettability. Our observations indicated that this mucus floats on the undersurface of the water after secretion from the foot, thereby enabling Lymnaea to use it as a "foothold" for upside-down gliding just beneath the water surface.

In attempting to examine how Lymnaea locomotes normally (i.e., "right-side-up"), we placed snails on either hydrophobic or hydrophilic plates and measured their speed. We did not find any significant difference in locomotory speed between the hydrophobic Teflon plates and the hydrophilic silicone plates. These results are interesting, as they show that the secretion with low wettability is useful for locomotion on any interface, including hydrophobic Teflon plates and hydrophilic silicone plates. When these snails glide upside down on the surface tension of the water, they do so using the mucus secreted from the foot and not the water molecules at the air-water interface.

A somewhat surprising result that emerged from our studies was that the upside-down gliding was faster than the normal right-side-up locomotion. The speed of upside-down locomotion on either plate (Teflon or silicone) was about 0.5 mm [s.sup.-1], and we had previously reported that snails loco-moting right-side-up in water had a speed that varied between 0.3 and 0.6 mm [s.sup.-1] (Wagatsuma et al., 2004). These latter speeds (in water and on the plates) are about 1/2 -1/3 of the speed of upside-down gliding (Fig. 2). We are at this point uncertain why upside-down gliding is faster than right-side-up locomotion.

We had hoped to be able to clarify the relationship between surface tension and upside-down gliding. To this end, we used two different surface-active agents, the detergent Tween 20 and ethanol, to decrease the surface tension of the water. We had thought that decreasing the surface tension would cause the snails to sink. However, sinking did not occur even when we used 100% Tween 20. Rather, the snails were repelled from the point of contact with the surface-active agent and remained attached to the underside of the surface. Although they often stopped gliding, particularly with ethanol application, they did not sink. Possibly, if we had applied these surface-acting agents more globally rather than just over the snail, we would have caused the snails to sink.

The mucus that is secreted from the snail's foot and that appears to be necessary for locomotion is generally made up of mucins, which are thought to be glycoproteins, and inorganic salts (Kimura et a!., 2003; Lincoln et al., 2004). It has proven difficult to characterize exactly what mucin is (Carlstedt et al., 1993). In Lymnaea, Ballance et al. (2004) have started the characterization of this mucus, reporting that it is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though they cannot rule out that polysaccharides may also be present.

We are still left with the question of how Lymnaea glides upside down. We suggest that this upside-down gliding is mainly due to a beating action of the cilia (Miller, 1974) and partially a peristaltic contraction of the foot muscles. The data obtained from the scanning electron microscope study showed that the diameter of individual cilia is on the order of a few hundreds of nanometers and that the cilia are not evenly distributed over the surface of the foot (Fig. 4). These findings regarding the distribution of cilia were confirmed by interference reflection microscopy (Fig. 5), Using this technique, we saw that some cilia formed dense isiand-like regions. It is these cilia that attach themselves to the secreted mucus, and it is by this means that the snails cling to the underside of the water. We also saw that most, but not all, of these cilia beat simultaneously in a similar direction (parallel to the direction of movement). We believe that the simultaneous beating of the cilia results in the driving force that enables the upside-down gliding behavior in Lymnaea. The remaining cilia are thought to be passively dragged along during the gliding. Peristaltic contractions of the foot muscles also play a role in the locomotory activity. The snails extend forward the anterior part of the foot powered by the beating cilia. Next, they use this part of the foot as it is attached to the secreted mucus by the cilia as an anchor. The snails then contract the posterior part of the foot. Such repeated extension and contraction of the foot in concert with the beating of the cilia allows forward movement.

In conclusion, Lymnaea is capable of active upside-down gliding at the underside of the water surface. The snails secrete mucus from the foot and use it as a foothold via the attachment of beating cilia for the upside-down gliding. The speed of upside-down gliding is determined in part by the time of day (the circadian component) and the temperature of the water.

Acknowledgments

This work was supported in part by grants-in-aid for scientific research (KAKENH1; no. 19370030) from the Japan Society for the Promotion of Science to E.I. and KAKENHI (nos. 18047003 and 18047004) for Scientific Research on a Priority Area, "Emergence of Adaptive Motor Function through Interaction between Body, Brain and Environment," from the Japanese Ministry of Education, Culture, Sports, Science and Technology to E.I. and K.G.

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KANAKO AONO (1),[dagger], AYACHIKA FUSADA (1),[dagger] YORICHIKA FUSADA (1),[dagger], WATARU ISHII (1),[dagger] YUJI KANAYA (1),[dagger], MAMI KOMURO (1),[dagger] KANAE MATSUI (1),[dagger] SATORU MEGURO (1),[dagger], AYUMI MTYAMAE (1),[dagger], YURIE MIYAMAE (1) [dagger], AYA MURATA (1) [dagger], SHTZUKA NARITA (1) [dagger] HIROE NOZAKA (1),[dagger] WAKANA SAITO (1),[dagger], AYUMI WATANABE(1),[dagger] KAORI NISHIKATA (1),[dagger] AKIRA KANAZAWA (2), YUTAKA FUJITO (3), MIKI YAMAGISHI (4), TAKASHI ABE (5), MASAFUMT NAGAYAMA (5). TSUTOMU UCHIDA (5), KAZUTOSHI GOHARA (5), KEN LUKOWIAK (6), AND ETSURO ITO (4)[double dagger], *

(1) Biology Club, Hokkaido Sapporo Okadama High School, 2-chome, Kitaokadama 1-jo, Higashi-ku, Sapporo 007-0881, Japon; (2) Biology Laboratory, Hokkaido Science Education Center, 7-chome, Miyanomori 4-jo, Chuo-ku, Sapporo 064-0954, Japan; (3) Department of Physiology, School of Medicine, Sapporo Medical University, Minami 1-jo, Nishi 17-chome, Chuo-ku, Sapporo 060-8556, Japan; Laboratory of Functional Biology, Kagawa School of Pharmaceaticat Sciences, Tokushima Bunri University, 1514-1 Shido, Sanuki 769-2193, Japan; (5)Division of Applied Physics, Graduate School of Engineering, Hokkaido University, Kita 13-jo, Nishi 8-chome, Kita-ku, Sapporo 060-8628, Japan; and 6Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada

Received 24 April 2008; accepted 9 July 2008.

* The preliminary results were presented at the 11th Symposium on Invertebrate Neurobiology organized by the International Society for In vertebrale Neurobiology in 2007, and thus some preliminary data were published in the proceedings: Aono, K. et al. 2008. Speed of back-su-swimming of Lymnaea. Acta Biologica Hungarica 59 (Suppl): 105-109.

[dagger] The student members of the Biology Club of Hokkaido Sapporo Okadama High School contributed equally to this work and are listed in alphabetieal order.

[double dagger] To whom correspondence should be addressed. E-mail: eito@kph.. bunri-u.ac.jp
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Author:Aono, Kanako; Fusada, Ayachika; Fusada, Yorichika; Ishii, Wataru; Kanaya, Yuji; Komuro, Mami; Matsui
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2008
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