Calcium ions in the aquatic environment drive planarians to food
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.
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.
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.
KeywordsPlanarian Environmental changes Quantitative behavioristics Feeding behavior Environment-responsive behavior Responsive sensitivity
Environmental changes affect homeostasis, growth, reproduction, and other essential biological functions, and animals sense environmental conditions and accordingly adopt survival strategies, such as activity, migration, reproductive behavior, and morphological alteration. Physiological end effects in animals, such as reproductive defects or behavioral disorders, caused by environmental factors such as pollutants or toxins have been intensively studied; however, how changes of natural environmental factors influence common animal behaviors, which are regulated by complex physiological and neural processes, remains poorly understood despite their importance for animal ethology, ecology, and evolution.
The planarian Dugesia japonica, a free-living flatworm, inhabits clear freshwater, such as rivers and lakes , and planarians are susceptible to toxins or pollutants in their aquatic environment. Therefore, planarians have been used as an indicator species in the fields of ecology and toxicology to assess the effect of water pollutants on aquatic animals [2, 3]. Moreover, planarians are well known for their remarkable regenerative ability [4, 5, 6]. Due to their unique physiological traits and phylogenetic position, planarians have been extensively used as experimental animals in the laboratory for investigations in a wide variety of biological fields, such as regenerative machinery, dynamics of stem cells, organogenesis, neuroscience, reproductive strategy, and animal evolution. In such investigations, various sustainable culture methods have been used, such as culturing in dechlorinated tap water , artificial culture solution [8, 9], or diluted artificial seawater [10, 11]. It was previously found that calcium, potassium, and sodium ions are important for culturing planarian cells and tissues [8, 12, 13], and remarkably, planarians can survive for a long time in a solution containing only calcium chloride , indicating that calcium ions are sufficient for planarian survival. In addition, lithium induces the formation of supernumerary eyes, one of the most evident malformations in planarians. The degree of this lithium-induced malformation is modulated by the competitive effects of sodium and potassium ions in the water in which planarians live . These findings suggest that ions in the environmental water may impact planarian physiology; however, how ions in the aquatic environment affect planarian behaviors subject to neural regulation remains obscure.
The central nervous system of planarians is composed of a bi-lobed brain in the anterior region of the animal and two longitudinal ventral nerve cords along the body [16, 17]. Recent molecular studies have demonstrated that, despite their simple body pattern, planarians have a sophisticated brain [18, 19]. Furthermore, studies using behavioral assays quantifying complex behaviors demonstrated that planarians display multiple environment-responsive behaviors mediated by the sensing of environmental signals, such as light, soluble chemicals, and temperature, via distinct sensory organs and integrating them in the brain [20, 21, 22, 23, 24]. Among their environment-responsive behaviors, planarians’ feeding is a complex behavior that can be divided into distinct processes. Planarians ingest food via the pharynx, which is protruded out from the middle portion of the body, after localization of the target food using soluble substances released from foods as the clue [25, 26]. Despite the variety of available quantitative behavioral assays [20, 21, 22, 23, 24], a method to quantify the food intake as the endpoint of the feeding behavior is still lacking. Recently, the usefulness of the visualization of colored chalks ingested together with food and double-stranded RNA for achieving stable RNAi gene knockdown in planarians was described , which suggests that feeding of colored chalks plus food may help to quantify the amount of food ingested. These findings indicate that behavioral assays in planarians offer unique opportunities for investigating animal behaviors associated with environmental changes. Here we investigated long-term and acute effects on planarian feeding behavior in aquatic environments with different ion compositions.
Highly purified water (pure water) (18 MΩ cm resistivity)(Merck), diluted artificial seawater (ASW) (Instant Ocean Sea Salt; Spectrum Brands), dechlorinated tap water (Bureau of Waterworks, Tokyo Metropolitan Government), and Kanatani’s planarian breeding water (hereafter referred to as “Kanatani water”) (0.77 mM CaCl2, 0.07 mM KCl, 0.64 mM NaCl, 0.17 mM NaHCO3, pH 7.6)  were used. The ion composition of modified Kanatani water used in this study is shown in Additional file 1: Table S1. ASW was prepared by dilution with pure water (Merck). Dechlorination of tap water was performed by autoclaving or treatment with sodium thiosulfate.
Ten planarians were cultured in a 90-mm-diameter plastic Petri dish containing 40 ml of a breeding water (Kanatani water, tap water, 0.05% ASW, 0.005% ASW, or 0% ASW/pure water) for 6 weeks. They were fed chicken liver twice a week and were cultured under conditions in which there was no competition for space or food, as referred to in previous reports [30, 31]. Breeding waters were changed every 2 days. Planarians that were 7-mm-long along the anterior-posterior axis and that had been derived from one culture tank and had been starved for 1 week were used as starting animals for behavioral experiments.
Assays of planarian behaviors
For the food-localization behavior assay, planarians were put in a 90-mm-diameter plastic Petri dish filled with test water, and a target food was put at the center of the dish. As a target food, a 5-mm cube of cut chicken liver or a food pellet was used. Planarian behavior was recorded for 5 or 10 min using a camera (Sony ILCE-7) fixed above the assay field. Trajectories were tracked every 200 msec, and the data were analyzed using a computer, Fiji/ImageJ, and R software v.3.5.1 (R Foundation for Statistical Computing, Vienna, Austria). The speed of an individual was calculated as the median value of the score measured every 200 msec during an assay.
For the photo-orientation behavior assay, planarians were put into a 60 × 30 × 10 mm container filled with test water . The container was exposed to 500 lx of white light emitted from a horizontally positioned light source and passed through a UV-cut filter. Planarian behavior was recorded for 90 s. The time animals spent in the target quadrant located opposite to the light source during the test was analyzed. The data were analyzed using the same method as used for the food-localization behavior assay.
To remove cilia of planarians, individuals were soaked in 1% ethanol in Kanatani water for 10 min at room temperature as previously described [36, 37], and subsequently planarians were used for behavioral assays in test waters, which contained no ethanol.
Live imaging of motile cilia
For live imaging of cilia in the peripheral epithelium, planarians were placed in a particular test water on a glass microscope slide attached to a perforated sheet of Parafilm and capped with a slide cover, as described previously [38, 39]. Movement of cilia was imaged by phase contrast microscopy on a Nikon Eclipse Ts2 microscope. Video segments were acquired at 240 frames/seconds and kymographs were produced from line scans perpendicular to the row of cilia using ImageJ/Fiji.
The ion composition of waters
For calculation of the concentration of calcium ions, potassium ions, and sodium ions in the Kanatani water (Additional file 1: Table S1), the tap water used in this study, and rivers in Japan, we used published data [8, 40, 41]. For selectively chelating calcium ions, ethylene glycol tetra-acetic acid (EGTA) was added at a final concentration of 10 mM to Kanatani water or tap water.
Data were analyzed by one-way analysis of variance (ANOVA), the Kruskal-Wallis test, or Dunnett test for overall group comparisons. For calculation of the difference from the normal distribution, the Kolmogorov-Smirnov test was used. The F-test was used to analyze normally distributed values. The statistical significance of differences between test results was determined by the Wilcoxon rank sum test or Welch two-sample t-test. Optionally, Holm correction was used to adjust significance levels for multiple comparisons. All tests were conducted using R software. Significance was defined as p < 0.05.
Breeding water affects the feeding behavior
To examine the proliferation of planarians as an outcome of nutrient intake, 10 individuals taken simultaneously from a population clonally cultured in a breeding tank were reared for 6 weeks with regular feedings in the respective breeding waters. The results shown in Fig. 1c indicate that the number of individuals increased monotonically to three (or more) times the starting number during 6 weeks of cultivation in tap water, Kanatani water, or 0.05% ASW in three independent batches; however, the number of individuals cultured in 0.005% ASW only approximately doubled during the 6-week period, and all of the planarians cultured in 0% ASW/pure water remained alive for 3 weeks but died without proliferation by 6 weeks (Fig. 1c). Planarians can generally survive for several months without feeding, and therefore the death of the planarians in 0% ASW/pure water might have been caused by the homeostatic state resulting from the extremely low concentration of salts in the breeding water, rather than by the loss of feeding. The doubling time (indicated by the horizontal dashed line in Fig. 1c) of individuals bred in Kanatani water or tap water was less than 2 weeks during the test. The doubling time of individuals bred in 0.005% ASW was 6 weeks, and increasing the concentration of ASW to 0.05% shortened the doubling time to 4 weeks. Statistical analysis at 6 weeks indicated that the population size was significantly different depending on the breeding water, and that the ion concentration in the breeding water affected the proliferation of planarians. These observations from breeding experiments suggest that reduction of the concentration of ions in the breeding water correlates with reduction of the food intake, resulting in a reduction of the population size of planarians.
To examine whether the low concentration or absence of ions in the breeding water specifically suppresses the feeding behavior, or whether it suppresses the overall behavioral capacities of planarians, we performed a photo-orientation assay using planarians after 2 weeks of cultivation in the respective breeding waters. Figure 2b shows the time spent in the target quadrant (the area opposite to the light source) during the photo-orientation assay using a rectangular assay field . Although planarians cultured in 0% ASW/pure water showed a reduction of light-avoidance behavior, we could not find any difference among the photo-orientation behavior of planarians cultured with a high concentration of ions in the breeding water (0.05% ASW), with an intermediate concentration of ions in the breeding water (0.025% ASW), and with a low concentration of ions in the breeding water (0.005% ASW) (Fig. 2b), and these values were comparable to those obtained in Kanatani water and tap water. Moreover, when we compared the speed of planarians’ movement in mm/sec, we could not find any difference among planarians cultured in Kanatani water, tap water, 0.05% ASW, 0.025% ASW, and 0.005% ASW, although impairment of the motor activity was observed in planarians cultured in 0% ASW/pure water (Fig. 2c). These results indicate that long-term breeding in the absence of any ions (0% ASW/pure water) reduces the motor activity of planarians, which might cause reduced scores in the feeding behavior and photo-orientation behavior assays; but the impairment of the feeding behavior found in planarians cultured in breeding water with a low concentration of ions (0.005% ASW) appeared not to be caused by reduced motor activity. These data indicate that the impairment of the overall behavioral activity of planarians in the absence of any ions (0% ASW/pure water) in the breeding water may have been caused by the resultant homeostatic state, whereas no clear impairment of overall behavioral activity was detected in planarians cultured in a low concentration of ions (0.005% ASW). Therefore, our data suggest that the feeding behavior was specifically inhibited by a low concentration of ions in the breeding water.
The environmental water immediately affects the feeding behavior
By contrast, the food intake differed depending on the environmental water used in the feeding behavior assay (Fig. 3d). Even planarians cultured long-term with a low concentration of ions (0.005% ASW) showed a higher score of food intake immediately after transfer into Kanatani water, tap water, or 0.05% ASW (Fig. 3d), and their scores were comparable to those of planarians cultured long-term in tap water, Kanatani water or breeding water with a higher concentration of ions (0.05% ASW) (Fig. 1b).
Meanwhile, we found that the food intake of planarians was increased in a food-dose-dependent manner (Fig. 3d). When we used food containing 62.5% liver homogenate, planarians tested in all environmental waters ingested the food. However, when we reduced the concentration of the liver homogenate in the target food to 40%, the feeding indexes of planarians in tap water, Kanatani water, and 0.05% ASW were drastically reduced, and planarians assayed in 0.005% ASW or 0% ASW/pure water failed to consume the food. Furthermore, the planarians tested in any environmental water did not ingest the food containing 33.4% liver homogenate. This result suggests that ions in the environmental water modulate the sensitivity of the responsiveness to foods that produces feeding behavior of planarians.
Calcium ions in the environmental water are required for the feeding behavior
In addition to using these modified defined waters during the food-intake assay, we devised a tracking assay to measure and quantify in more detail the food-localization behavior, as shown in Fig. 5c. The results of the food-localization assay shown as the averaged movements of 10 independent individuals together with a heat map for these movements indicated that planarians tested in Kanatani water tended to move toward the quadrant where target food was located, indicated by a white cross, which was consistent with the above results of the food-intake assay (Fig. 5c). However, planarians assayed in the Kanatani water lacking calcium ions showed defects in their ability to move toward the food, and instead showed random movements around the start region. In contrast, planarians tested in the Kanatani water without potassium or sodium ions moved toward the food, like planarians in the original Kanatani water. Quantification of the time spent in the target quadrant by these animals clearly indicated that calcium ions, but not potassium or sodium ions, in the environmental water are required for the food-localization behavior (Fig. 5d).
In order to confirm that this requirement for calcium ions did not arise from an effect on the speed of movement, we compared the speed of movement among planarians in the respective waters. The results showed that the absence of calcium ions in the environmental water did in fact slightly impair the speed of movement (Fig. 5e). Therefore, we further evaluated the time spent in the target quadrant as the food-localization index, calculated by assuming that all individuals had the same speed by compensating with the median value of the speed (0.38 mm/sec) of the planarians in the Kanatani water in the absence of calcium ions. The compensated value of food-localization behavior expressed as the food-localization index showed that absence of calcium ions in the environmental water during the assay reduced the activity of food-localization behavior, whereas the absence of potassium or sodium ions did not affect the food-localization behavior (Fig. 5f). These results suggest that absence of calcium ions in the environmental water also reduces motor activity; however, compensating for the reduced motor activity showed that the low score of the time planarians spent in the target quadrant appeared not to be caused by the decrease of the speed due to the absence of calcium ions in the environmental water. Consistent with these results, the ability to move toward the target food indicated by the food-localization index and the food intake indicated by the feeding index were impaired in planarians assayed in Kanatani water or tap water supplemented with EGTA to chelate calcium ions (Fig. 5g-i, Additional file 2: Figure S1).
Planarians exhibit a gliding mode of movement using motile cilia in the peripheral epithelium throughout the body, as well as a crawling mode of movement using muscles, during locomotion. In order to investigate whether the absence of calcium ions in the environmental water affects ciliary beating, we observed ciliary beating in the peripheral epithelium in each environmental water tested here, and could not find any defect of the beating in any of the waters (Additional file 3: Figure S2a). Furthermore, we performed the food-localization assay using planarians whose cilia were removed by treatment with 1% ethanol, and the results showed that the absence of cilia did not affect the activity of moving toward the food, although the speed of moving was slowed due to the loss of gliding ability (Additional file 3: Figure S2b-f). These findings indicated that the absence of calcium ions in the environmental water does not affect the ciliary beating, and motile cilia are not required for the activity of producing the feeding behavior. Taken together, our data suggest that calcium ions in the environmental water were required for the feeding behavior independent on the requirement for calcium for muscle activity in planarians.
Calcium ions in the environmental water boost the feeding activity
Here we showed that the sensitivity of the responsiveness to foods in planarians is modulated by calcium ions in the environmental water. In insects, octopaminergic neurons and dopaminergic neurons in the central nervous system regulate the sensitivity to foods during feeding behavior [42, 43, 44, 45, 46, 47], and in the neurons that respond to the respective amines, calcium ions, which act as a second messenger, are increased in the cytosol as a result of activation of a guanine nucleotide-binding protein (G protein) . These findings suggest that deficiency of calcium in the environmental water may interfere with the activity of octopaminergic or dopaminergic neurons in the brain that regulate the feeding behavior of planarians. However, we found here that the removal of calcium ions from the environmental water immediately impaired the feeding behavior (Figs. 5, 6), suggesting that calcium ions in the environmental water are involved in the activation of the chemosensory neurons distributed in the body surface rather than brain neurons.
It has been reported that the feeding behavior of planarians occurs by sequential processes consisting of food-localization behavior and food-intake behavior by the pharynx protruded from the body . Our data in the present study showed that deficiency of calcium ions in the environmental water inhibited both the food-localization and food-intake processes (Figs. 5, 6). These results suggest that similar calcium-concentration-dependent signaling machinery may be involved in these behavioral processes determining the sensitivity of the response to foods. Transient receptor potential (TRP) channels known to play critical roles in the transduction of multimodal sensory signals, including olfaction, are non-selective cationic channels, which allow for the influx of calcium ions into cells. In planarians, some TRP channel genes were identified and were found to be widely distributed in the periphery of the body and in various tissues [21, 49, 50], although no TRP channel genes expressed in chemosensory neurons in lateral branches elongated from the brain or in the pharynx in the middle of the body have been identified. These findings collectively suggest that the sensitivity of the chemosensory signaling coupled with transducers such as TRP channels may be influenced by calcium ions in the environmental water in a concentration-dependent manner, resulting in modulation of the feeding behavior in planarians.
Additionally, while planarians in 0% ASW/pure water displayed photo-orientation behavior with normal movement activity (Fig. 3b, c), planarians in modified Kanatani water lacking calcium ions showed reduced movement activity (Fig. 5d, e), suggesting that the balance of various ions, rather than calcium ions alone, may be important for producing planarians’ behaviors. By combining further data regarding behavioral traits obtained from the kinds of experiments presented here and additional data regarding molecules in the neurons regulating behaviors, we should be able to unravel the relationship between the neural system and ions in the environmental water in planarians.
Recently planarian biology has been advanced as a model system for neuroscience, as well as for regeneration and stem cell biology. However, the ecology of planarians in nature remains poorly investigated despite the importance of understanding animal physiology in particular environments, as well as its importance for determining appropriate culture conditions in the laboratory. In contrast to the dramatic effects of pollutants or toxins on planarian survival and general behaviors, the chemical properties of habitat water have been thought to have only a minor impact on planarian survival or populations . In this study, we found that the population growth rate of planarians differs depending on the culture water. This observation, combined with the data of behavioral analyses, suggests that chemosensory neurons closely associated with the feeding behavior may be modulated by calcium ions in the environmental water, with resultant impacts on the growth and reproduction of planarians. These insights have implications about ethological and ecological effects that might result from differences of the concentration of calcium ions in planarians’ habitat waters, including possible restrictions of planarians’ distribution range in nature. Indeed, previous reports indicated that although planarians can survive in lakes with low levels of dissolved calcium ion, planarians tended to be abundant in lakes with high levels of this ion [52, 53].
Planarians are postulated to have originated in Gondwanaland (Africa) approximately 300 million years ago and then spread to southern Europe and eventually to have reached the Far East [54, 55]. Therefore, planarians may have initially been optimized for a habitat with a higher concentration of calcium ions, and may have adapted to lower concentrations of calcium ions as their distribution became wider, and thus the boosting of the feeding behavior by calcium ions might be a result of maintaining the ancestral phenotype. Consistent with this, the feeding behavior in S. mediterranea, which dwells in hard water, was also improved by excess administration of calcium ions, like that in D. japonica (Fig. 6).
Although the acidification of surface-waters and rain has been mitigated by environmental regulations and agreements since the 1990s, the concentration of calcium ions was increased worldwide by acid deposition in the 1960s and 70s [56, 57]. Planarians are known as a top predator in their habitats ; therefore, our results imply that increases of calcium ions in aqueous systems may contribute to the extinction or decline of particular animals due to excessive predation by planarians. Taken together, the present experimental findings provide insights into not only planarians’ behavioral traits but also possible ecological impacts.
We demonstrated that breeding water ionic properties specifically impact feeding behavior in planarians, and the feeding behavior was suppressed when the concentration of ions in the breeding water was low, and eventually affected the population size. Quantitative analyses of the feeding behavior immediately after transferring the planarians to different environmental waters clearly showed that feeding behavior was affected by the concentrations of ions in the environmental water itself, rather than that some defect of the homeostatic state during long-term culture in breeding water affects the feeding behavior. Using defined waters, we identified calcium ions in the environmental water as ions indispensable for the feeding behavior, as well as for potentiating it via affecting the sensitivity of the responsiveness to foods. Furthermore, our results together with comparison between the chemical compositions of the laboratory breeding waters used in this study and of bodies of water in nature suggested that planarians might potentially affect biodiversity by changing the activity of their feeding behavior according to the chemical composition of the environmental water. We propose that even slight changes in environmental factors in nature may impact the ecosystem via modification of animal behavior.
We thank Dr. Yoshihiko Umesono, and Dr. Norito Shibata for their critical reading of the manuscript and suggestions. We also thank Dr. Elizabeth Nakajima for critical reading of the manuscript, and all of our other laboratory members for their help and encouragement.
MM and TI conducted the behavior analyses and data analysis. MN performed data analysis. MI, NK, YS, and RB performed the breeding experiments. KA supervised the project. TI conceived and designed the project, and wrote the manuscript. TH and KA edited the manuscript. All authors read and approved the final manuscript.
This work was supported by Grants-in-Aid for Scientific Research (15 K07148, 18 K06341) to TI, The Takeda Science Foundation fund to TI, and a Grant-in-Aid for Scientific Research (16H06376) to KA.
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The authors declare no competing financial or non-financial Interests.
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