Calcium ions in the aquatic environment drive planarians to food

Abstract

Background: Even subtle changes in environmental factors can exert behavioral effects on creatures, which may alter interspecific interactions and eventually affect the ecosystem. However, how changes in environmental factors impact complex behaviors regulated by neural processes is largely unknown. The freshwater planarian Dugesia japonica, a free-living flatworm, displays distinct behavioral traits mediated by sensitive perception of environmental cues. Planarians are thus useful organisms for examining interactions between environmental changes and specific behaviors of animals.

Results: Here we found that feeding behavior was suppressed when the concentration of ions in the breeding water was low, while other behaviors were unaffected, resulting in differences in population size. Notably, the decline in feeding behavior was reversed in an ion-concentration-dependent manner soon after the planarians were moved to ion-containing water, which suggests that ions in environmental water rapidly promote feeding behavior in planarians. Moreover, the concentration of ions in the environmental water affected the feeding behavior by modulating the sensitivity of the response to foods. Finally, we found that calcium ions in the aquatic environment were required for the feeding behavior, and exposure to higher levels of calcium ions enhanced the feeding behavior, showing that there was a good correlation between the concentration of calcium ions and the responsiveness of planarians to foods.

Conclusions: Environmental calcium ions are indispensable for and potentiate the activity level of the feeding behavior of planarians. Our findings suggest that the ions in the aquatic environment profoundly impact the growth and survival of aquatic animals via modulating their neural activities and behaviors.

Keywords: Environment-responsive behavior; Environmental changes; Feeding behavior; Planarian; Quantitative behavioristics; Responsive sensitivity.

Fig. 1

Breeding water affects food intake and population size. a. Typical living animals that had been cultured in five kinds of breeding water and had then ingested liver homogenate tagged with a pink powder. Ingested food was visualized using a Texas Red fluorescence filter set. No fluorescence signals were detected in the unfed planarians. The strong fluorescence signals were detected in the planarians grown in Kanatani water, tap water, or 0.05% ASW, while weak signals were detected in planarians grown in 0.005% ASW or 0% ASW/pure water. Anterior is to the left. Bar, 1 mm. All animals are shown from the dorsal side. b. The feeding index determined by the ratio between the fluorescence area and the whole body area is shown as a box-and-whisker plot with mean (open circles). The feeding index of planarians grown in Kanatani water, tap water, or 0.05% ASW showed high score and there were no significant differences (NS by Kruskal-Wallis test) among them. By contrast, a low concentration (0.005% ASW) or absence (0% ASW/pure water) of ions in the breeding water reduced the score of the feeding index. **, p < 0.01 (Wilcoxon test). c. Population growth curve of planarians cultured in breeding waters for 6 weeks. The curves are shown as a trend line of three independent batches with 95% confidence interval (gray). The horizontal dashed line indicates the doubling of the number of individuals present at the start point. The number of individuals at 6 weeks was different, depending on the breeding water. ***, p < 0.005 (ANOVA)

Fig. 2

Breeding water affects the food-localization behavior. a. The percent of individuals that reached the food (piece of liver) within 10 min is shown as a box-and-whisker plot with mean (open circles). n = 10; 4 independent experiments. Although there were no significant differences (NS by Kruskal-Wallis test) among planarians grown in Kanatani water, tap water, 0.05% ASW, and 0.025% ASW (Kruskal-Wallis test), the food-localization of planarians cultured in breeding water containing a lower concentration (0.005% ASW) or absence (0% ASW/pure water) of ions was suppressed. *, p < 0.05 (Wilcoxon test). b. The time spent in the target quadrant during the photo-orientation assay. Only the planarians that were grown in the 0% ASW/pure water showed impairment of the photo-orientation behavior, while the others were unaffected. There were no significant differences (NS) among planarians grown in Kanatani water, tap water, 0.05% ASW, 0.025% ASW, and 0.005% ASW (Kruskal-Wallis test). c. The motor activity indicated by the average speed. Although the speed of planarians grown in the pure water was slightly decreased, the others were unaffected. There were no significant differences (NS) among the speeds of planarians grown in Kanatani water, tap water, 0.05% ASW, 0.025% ASW, and 0.005% ASW (Kruskal-Wallis test)

Fig. 3

Environmental water immediately affects and can restore feeding behavior specifically. a. A schematic illustration of the experimental design for behavioral assays in various environmental waters. Planarians grown in 0.005% ASW for at least 2 weeks were transferred to assay fields containing each type of environmental water and briefly subjected to behavioral assays. b. The percent time spent in the target quadrant during 90 s in the photo-orientation assay is shown as a box-and-whisker plot with mean (circles). c. Speed of movement (mm/sec) during photo-orientation assay. There were no significant differences (NS) in either the photo-orientation behavior or the motor activity among planarians bred in the different environmental waters (Kruskal-Wallis test). d. The concentration of ions in the environmental water determines planarians’ sensitivity to the presence of food. The food intake was analyzed using colored food pellets containing different proportions of liver homogenate (33.3, 40, and 62.5%). Planarians cultured long-term with a low concentration of ions (0.005% ASW) showed restoration of the feeding behavior immediately after transfer into Kanatani water, tap water, or 0.05% ASW. The feeding index increased in a food-proportion-dependent manner. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Wilcoxon test)

Fig. 4

The environmental water affects the sensitivity of the response to foods. a. The percent of individuals that reached the food within 10 min. Although the food-localization behavior of planarians tested in Kanatani water, tap water, or 0.05% ASW showed a high score, the scores of the food-localization were gradually decreased according to the reduction of the concentration of ions in the environmental water. Regression analysis showed a good correlation between the food-localization behavior and the concentration of the ions. n = 10; 3 independent experiments. b. The percent of individuals that reached the food within 30 or 60 min. Prolongation of the assay time resulted in improvement of the score of the food-localization behavior, indicating that low concentrations of ions in the environmental water may reduce the ability to elicit the food-localization behavior

Fig. 5

Calcium ions are required for the feeding behavior. a. Schematic illustration of the experimental design for the behavioral assays in environmental waters in the absence of a particular ion. Planarians grown in 0.005% ASW for at least 2 weeks were transferred to assay fields containing each type of modified Kanatani water and briefly subjected to behavioral assays. b. The feeding index of planarians in Kanatani water in the absence of a particular ion is shown as a box-and-whisker plot with means (circles). The absence of calcium ions in the environmental water reduced food intake, whereas the absence of potassium ions or sodium ions did not affect it. c. The food-localization assay in each type of modified Kanatani water. The cross indicates the position of the colored food pellet. The circle indicates the planarian’s start region. Planarian behavior was quantified using the time spent in the target quadrant. The diameter of the circular field was 9 cm. Heat map view of the averaged behavior of 10 individually assayed animals in a food-localization assay field. Warm colors indicate locations where much time was spent, and cool colors those where little time was spent. Planarians in Kanatani water, Kanatani water without potassium ions, or Kanatani water without sodium ions showed a preference for moving to and remaining in the region with the food, whereas planarians in Kanatani water without calcium ions did not show such food-localization behavior. t = 300 s. d. Time spent in the target quadrant during assay of planarians in Kanatani water without a particular ion is shown as a box-and-whisker plot with mean (circles). e. Speed of movement of planarians during the assay. The absence of calcium ions in the environmental water impaired the motor activity. f. The food-localization index is the adjusted value of the spent time in the target quadrant calculated by assuming all individuals had the same speed. The adjustment was performed using the median value of the speed (0.38 mm/sec) of the planarians in Kanatani water without calcium ions. g. The food-localization assay in Kanatani water containing 10 mM EGTA. Planarians in Kanatani water (Ctrl) showed a preference for moving to and staying in the region with the food, whereas planarians in Kanatani water containing EGTA did not show such food-localization behavior. t = 300 s. h. The food-localization index is the adjusted value of the spent time in the target quadrant calculated by assuming all individuals had the same speed of movement. The adjustment was performed using the median value of the speed (0.51 mm/sec) of the planarians in Kanatani water containing EGTA. i. The feeding index of planarians in Kanatani water containing EGTA is shown as a box-and-whisker plot with means (circles). The chelation of calcium ions by EGTA in the Kanatani water reduced the food intake. *, p < 0.05; **, p < 0.01; ***, p < 0.005; NS, not significant (Wilcoxon test)

Fig. 6

Calcium ions are required for and improve food intake. a. Feeding indexes of D. japonica in Kanatani water lacking calcium ions (Ca⁺⁺ (−)), Kanatani water containing a low concentration of calcium ions (0.1x Ca⁺⁺), original Kanatani water (1x Ca⁺⁺) or Kanatani water containing excess calcium ions (10x Ca⁺⁺). b. Feeding indexes of S. mediterranea under the same conditions as tested in D. japonica. n = 10; NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Wilcoxon test)

Fig. 7

Comparison of ion concentrations among rivers in Japan and water characteristics used in this study. Concentrations of calcium, potassium, and sodium ions in rivers throughout Japan are indicated by gray dots. Concentrations of calcium, potassium, and sodium ions of tap water and Kanatani water are indicated by red dots. Both the tap water and Kanatani water contain a relatively high concentration of ions compared to those of rivers in Japan. Circles and vertical bars are mean ± sd