Revisiting Chirality in Slime Mold: On the Emergence and Absence of Lateralized Movement in Physarum polycephalum Influenced by Various Stimuli

Abstract

Behavioral lateralization in animals is a well-known phenomenon; however, it has only rarely been studied in unicellular organisms. A groundbreaking study found lateralized movement in T-mazes in the formless plasmodia of the slime mold Physarum polycephalum. In this work, a replication of that study was conducted in a specially designed, elaborated T-maze system. Considering the amoeboid organism’s diverse sensory capabilities, we further comprehensively investigated the influence of light, artificial magnetic fields, the magnetic field of the Earth, and vibration on movement direction. Two different clonal lines were tested to assess genetic diversity, encompassing over 1600 individual plasmodia. Our results show that no general lateralized behavior exists in the absence of stimuli in both clonal lines. On the other hand, Physarum’s sensitivity to strong magnetic fields and vibration induces significant true lateralization in previously nonlateralized plasmodia (37.6% right and 62.4% left, respectively). Possible mechanisms behind this induced lateralization are discussed. We conclude that previous findings showing lateralization are likely to have been influenced by unknown external stimuli.

1. Introduction

While most animals appear bilaterally symmetric on the outside, asymmetry can be found in substructures of any size, from the organization of the human brain to neuronal connections and even on the molecular level. The underlying asymmetry of the nervous systems often creates lateralized behavior in the organism, from handedness in humans to differences in perception and processing of stimuli [1]. These findings are widespread among both vertebrates and invertebrates [2]. Therefore, it has been theorized that lateralization arose early in evolution and conveys an evolutionary advantage to organisms [1].

The reason for the development of asymmetry is still unknown, but it is theorized to occur early in embryonic development. While various theories about the exact reason for the development of left–right patterning exist, they typically involve cellular mechanisms causing and/or upholding the break in symmetry [3]. In multicellular organisms, ranging from plants to nematodes, frogs, and even humans, the cytoskeletal component tubulin and associated proteins are considered the origin of asymmetry [4]. Additionally, a variety of other mechanisms play an important role in the development of asymmetry in some organisms, such as chromatic segregation [5] and the activity of cilia [3].

Due to these cellular mechanisms, asymmetry is, therefore, not only relevant for multicellular organisms but can be found in single cells of metazoan bodies and unicellular organisms as well.

While morphological asymmetry in unicellular organisms has been studied, so far, few studies on lateralized movement and behavior exist. The growing trend in basal cognition, the analysis of behavior and learning in aneural organisms, gave rise to a growing number of behavioral experiments on unicellular organisms. These show a wide variety of adaptive mechanisms in multiple species and allow for theorizing about the underlying mechanisms of cellular cognition [6]. But, since cellular lateralization seems to be caused by an evolutionarily conserved mechanism found in both plants and animals [7], it is possible that the same mechanism has already been active in the single-celled ancestor of both, and, therefore, also their other protist relatives. To gain a deeper understanding of basal cognition and cell behavior, behavioral lateralization as a possible influence mechanism has to be considered.

The first experiment on behavioral lateralization in a single-celled organism was performed in Physarum polycephalum [8]. The true slime mold Physarum polycephalum is a unicellular amoeboid organism that consists of a multinucleated cell called a plasmodium. Plasmodium can actively move and reorganize itself by extending or retracting pseudopodia, allowing for speeds up to 5 cm per hour [9]. They can grow to 900 cm², making them easily observable to the naked eye and allowing for a wide variety of behavioral observations [10]. Despite being a single-celled amoeba, Physarum has been shown to be capable of finding the shortest paths through mazes [11], solving complex decision-making paradigms [12], and even being capable of habituation to aversive stimuli [13]. It is an important model for research in both aneural intelligence and cell physiology.

In an experiment by Dimonte et al. [8], small pieces of Physarum were placed in T-mazes. Forced to make a choice, 76% of the plasmodia chose a right turn, with the rest choosing either a left turn or branching into both directions. While the authors state that the mechanism behind laterality in Physarum remains unknown, they speculate that chiral intracellular structures might play a role in the lateralized movement pattern.

This finding is important because, unlike lateralized animals, Physarum does not possess a fixed bilaterally symmetrical shape but instead a formless, constantly shifting mass of pseudopodia. It is, however, consistent with the few findings on intracellular handedness in other unicellular organisms [14] and learned turning preferences of sperm cells in T-mazes [15]. Furthermore, while Physarum itself has no fixed shape, it is capable of linear movement through the formation of tubuli, long, vein-like structures for the transport of endoplasm. The formation and self-organization of these structures give the plasmodium a temporary shape that, while not symmetrical, possesses one or more temporary front-and-back ends, not unlike bilaterally symmetrical animals.

To gain insight into the mechanism behind slime mold lateralization and as a prerequisite for further behavioral experiments, it is vital to look deeper into this phenomenon.

Physarum is capable of sensing a wide variety of stimuli. Like most unicellular organisms, they respond mainly to chemical stimuli but are far from limited to that. They exhibit both positive and negative phototaxis [16], seek to maintain an optimal temperature [17], and tend to move toward the direction of gravity [18]. It has also been found that Physarum, when kept in a magnetic field, tends to move within the magnetic field lines. This response, however, seems to be dependent on both the strength of the magnetic field and the size of the plasmodium [19]. Additionally, it has been shown in related species that even within populations, slime molds show high genetic diversity [20].

This study aims to replicate the main findings of Dimonte et al. [8], further comprehensively assessing how light, magnetic fields, vibration, gravity, and genotype influence lateralized behavior, thus providing new important insights into the behavioral complexity of Physarum.

2. Materials and Methods

2.1. Biological Material

The slime molds used in our experiments were purchased as dried sclerotia of unknown age from a non-scientific commercial provider who collected the original plasmodia in forests. The species was confirmed by inducing sporulation and subsequent analysis of the spores/sporocarp. Due to the fast growth and regular division of plasmodia and their genetic uniformity, our slime molds were clones grown from a single plasmodium. To control whether laterality might be different among populations, two different clone lines of Physarum polycephalum were used in the replication: one collected in Germany and another collected in France. After sporulation, both were identified as Physarum polycephalum, despite presenting different phenotypes in their plasmodial stage. The German line was lighter in color and tended to produce thicker, branching pseudopodia, while the French line moved in more interconnected, fan-like wave structures (Figure 1). These morphological differences remained stable over months of varied conditions and can, therefore, be seen as an indicator of different genotypes in different populations of the same species.

Routinely, the plasmodia were kept in darkness at 20 °C in closed 12 × 12 cm petri dishes on a nutrition-free 2% water–agar (20 g Agar Agar (Carl ROTH GmbH, Karlsruhe, Germany) per 1000 mL tap water) under clean but non-sterile conditions. They were fed dried oat flakes sprinkled on the plates and resettled to fresh plates every 2–3 days, supplied with fresh food, and sprayed with de-mineralized water to raise the humidity, only coming in contact with light during maintenance for up to 30 min every 2–3 days. The plasmodia were kept in a cupboard without notable sources of vibration or electrical equipment in the vicinity. Both clonal lines were maintained at the same time using the same protocol.

Pre-tests were conducted to determine the proper cell mass for the experiments. Since Physarum is capable of lateral splitting and spreading both ways in a T-maze, different cell masses were tested to determine a cell size incapable of splitting. Beginning with 20 mg, the mass was successively reduced to 1 mg, which was deemed both still viable for survival but only rarely led to splitting, although splitting and failure to survive occasionally occurred. Therefore, in the main experiments, all slime molds had an initial cell mass of circa 1 mg. Inoculation of the labyrinths was performed by cutting small pieces from the active moving fronts of plasmodia from the permanent culture, weighing the pieces, adjusting the weight if necessary, and then placing them in the labyrinth. A total of 1600 plasmodium fragments were tested across the six experiments, with over 200 fragments in each experimental setting.

2.2. Experiments

Seven series of tests were conducted in custom-designed and 3D-printed T-maze units comprising eight individual mazes (Figure 2). The individual mazes featured a vertical corridor 5 cm long and 0.5 cm wide, with two arms 2 cm long. Eight individual mazes were combined to form a complex in which two mazes each corresponded to a cardinal point.

This design allowed for control of external influences, as well as the influence of Earth’s magnetic field, as one side of the maze complex was aligned to the north using a compass. The bottom of the mazes was covered in water–agar without nutrients or other additives (20 g Agar Agar (Carl ROTH GmbH, Karlsruhe, Germany) per 1000 mL of tap water) to supply water to the plasmodia, the same medium in which the plasmodia were maintained outside the experiments. Between experiments, the labyrinths were cleaned with hot water and then prepared by filling the labyrinths with the boiled agar mixture using a glass pipette. While Dimonte et al. used filter paper as well, they found no significant difference between these two media [8]; therefore, in our experiment, agar was used throughout. These maze complexes were placed in square Petri dishes filled with water to keep humidity constant. For recording purposes, the mazes were placed on an LED lighting plate, illuminating the mazes from below with diffuse, white light at a low intensity of 200 Lux. This allowed for constant filming of the slime molds via a document camera at a rate of one picture every ten minutes, allowing for constant documentation without disturbing the slime mold. From the literature, it is known that this light intensity and spectrum do not influence the slime mold [21]. This experimental setup was placed in a closed wooden box for the duration of each experiment to keep out external light and other external influences; the box itself was kept in a room where the temperature was kept constant at 20 °C by constant air conditioning. To minimize the influence of electromagnetic fields and vibration, the experimental box was placed on a separate table, with the recording PC on another table about 70 cm away to the east of the experimental box. Every individual plasmodium was tested for 48 h, allowing most of them to fully move through the mazes.

The 7 different experimental setups are illustrated in Figure 3, and consist of the following variants:

1. Replication of published experiments. Experiment 1 was conducted under the described conditions without any additional stimuli in order to replicate the experiment by Dimonte et al. [8]. For this experiment and for the third and all subsequent experiments, clones of a plasmodium collected in Germany were used. For Experiment 1, 256 plasmodia were used;

2. Genotype influence. To control the influence of the genotype on behavioral lateralization, for the second experiment, a clone line collected in France was used. Otherwise, the experimental conditions were kept the same as in Experiment 1. For Experiment 2, 480 plasmodia were used, with a higher dropout rate due to their slower movement rate;

3. Phototaxis. Experiment 3 was used to explore the influence of light on laterality. An LED light-producing white light was affixed to the north side of the box at a height of 26 cm and indexed a light gradient from 4000 to 2000 Lux. For the first row of maze complexes, the incident angle was 55° measured in the middle of the complex. Second-row maze complexes were illuminated at an incident angle of 40°. Due to the different angles, the resulting shadow patterns were dissimilar, and the mazes were evaluated separately. For Experiment 3, 256 plasmodia were used;

4. Magnetotaxis. Experiments 4 and 5 aimed to further explore the influence of magnetic fields on Physarum behavior. Shirakawa et al. have previously shown that Physarum movement tends to align with magnetic field lines under certain conditions [19]. For Experiment 4, an artificial magnetic field was created utilizing 160 neodymium magnets (N48, axial magnetization, 3 mm diameter with a layer of Ni-Cu-Ni), which were placed on the north and south sides of the T-mazes. Due to the decline in magnetic strength, a gradient with a minimum of zero and a maximum of 3 mT formed, as measured with a gaussmeter. T-mazes positioned in the middle of the setting were separately evaluated as a group with a low magnetic field of 0.3–0.5 mT, and the margin mazes as a group with a moderate magnetic field from 2.5 to 3 mT. For Experiment 4, 320 plasmodia were used;

5. Inclination. Experiment 5 investigated the influence of the Earth’s magnetic field. While the maze complexes were already aligned with the cardinal directions, with one-fourth of all mazes facing directly north, the angle at which the magnetic field lines of the Earth intersect the surface varies between the equator and the poles. At the location of the lab in Northern Germany, the inclination angle is 69°. To control for this, a wedge was inserted to allow for the placement of the maze complexes at a 69° inclination angle. The wedge was oriented toward the north (declining) and south (ascending). For Experiment 5, 256 plasmodia were used;

6. Gravitaxis. While the setup of Experiment 5 allowed for better alignment with Earth’s magnetic field, it also did not allow for the separate movement toward north from movement downward. Since Physarum is known to exhibit gravitaxis, a sixth experiment was conducted to control for the effects of gravity. For this purpose, the wedge used in Experiment 5 was rotated by 180° to maintain the same angle toward the ground but to separate the effect of gravity on movement from the effect of magnetic field lines. For Experiment 6, 352 plasmodia were used;

7. Vibration. For Experiment 7, the same setup was used as in Experiments 1 and 2. Additionally, in the west, next to the experimental box, a vibration motor ran permanently at 300 Hz. To keep the motor from moving erratically and to instead distribute the vibration equally, the motor was encased in agar, creating a vibration propagating on the same table as the experiment. Previous observations in our lab suggest that Physarum reacts to vibration stimuli. For Experiment 7, 320 plasmodia were used.

2.3. Data Analysis

Video material was evaluated manually by the authors and coded according to a predefined scheme. Included were the decisions for only one arm, while lateral splitting into both arms was excluded (16.1% among all experiments). A trial was deemed finished when the plasmodium reached at least half an arm’s length. For all experiments, p-values were calculated using two-tailed binomial tests with a 0.5 success value. Due to the high number of tests performed, the Benjamini–Hochberg procedure for controlling the false discovery rate was conducted.

3. Results

The results for all experiments, grouped by decision between left and right, can be found in

Table 1. Observed left/right-movement choices of Physarum under various conditions, both as a total for each condition, as well as separated by starting cardinal directions. p-values were obtained using two-tailed binomial tests; values below 0.05 are shown in bold and marked with (*). p-values below 0.01 were deemed still significant after the Benjamini–Hochberg procedure and are marked with (**).

		Total		Start North		Start South		Start West		Start East
Experiment		Right	Left	Right	Left	Right	Left	Right	Left	Right	Left
1 Replication	n	121	122	34	30	29	28	36	25	22	39
	%	49.8	50.2	53.1	46.9	50.9	49.1	59	41	36.1	63.9
	p	1		0.532		0.792		0.124		0.040 *
2 Genetic variation	n	109	116	27	24	21	33	32	28	29	31
	%	48.4	51.6	52.9	47.1	38.9	61.1	53.3	46.7	48.3	51.7
	p	0.690		0.576		0.134		0.518		0.898
3 Phototaxis	n	106	106	28	28	33	19	21	32	24	27
(total)	%	50	50	50	50	63.5	36.5	39.6	60.4	47.1	52.9
	p	0.946		0.894		0.036 *		0.168		0.780
3a Phototaxis	n	58	53	12	15	17	11	15	13	14	14
(4000 lux)	%	52.3	47.7	44.4	55.6	60.7	39.3	53.6	46.4	50	50
	p	0.570		0.702		0.184		0.572		0.850
3b Phototaxis	n	48	53	16	13	16	8	6	19	10	13
(2000 lux)	%	47.5	52.5	55.2	44.8	66.7	33.3	24	76	43.5	56.5
	p	0.690		0.458		0.064		0.014 *		0.678
4 Magnetotaxis	n	134	143	37	33	36	36	24	42	37	32
(total)	%	48.4	51.6	52.9	47.1	50	50	36.4	63.6	53.6	46.4
	p	0.630		0.550		0.906		0.036 *		0.470
4a Magnetotaxis	n	58	86	13	24	17	21	14	18	14	23
(2.5–3 mT)	%	40.3	59.7	35.1	64.9	44.7	55.3	43.8	56.3	37.8	62.2
	p	0.024 *		0.098		0.628		0.596		0.188
4b Magnetotaxis	n	76	57	24	9	19	15	10	24	23	9
(0.3–0.5 mT)	%	57.1	42.9	72.7	27.3	55.9	44.1	29.4	70.6	71.9	28.1
	p	0.082		0.004 **		0.392		0.024 *		0.008 **
5 Inclination	n	110	101	27	29	31	23	35	17	17	32
	%	52.1	47.9	48.2	51.8	57.4	42.6	67.3	32.7	34.7	65.3
	p	0.492		0.894		0.220		0.008 **		0.044 *
6 Reverse Inclination	n	99	131	24	22	24	41	9	49	42	19
	%	43	57	52.2	47.8	36.9	63.1	15.5	84.5	68.9	31.1
	p	0.040 *		0.658		0.046 *		<0.001 **		0.002 **
7 Vibration	n	76	126	16	35	32	17	16	36	12	38
	%	37.6	62.4	31.4	68.6	65.3	34.7	30.8	69.2	24	76
	p	0.001 **		0.011 *		0.0212 *		0.008 **		<0.001 **

.

Table 2. Observed movement for each experiment classified by choice for cardinal directions. Depending on the starting point, each Physarum faced a choice between cardinal directions. For example, a plasmodium starting toward west would have to choose between moving north or south. p-values were obtained using two-tailed binomial tests. p-values below 0.01 were deemed still significant after the Benjamini–Hochberg procedure and are shown in bold and marked with (**).

Experiment		North	South	East	West
1 Replication	n	75	47	62	59
	%	0.61	0.39	0.51	0.49
	p	0.008 **		0.716
2 Genetic Variation	n	63	57	60	45
	%	0.53	0.48	0.57	0.43
	p	0.522		0.118
3 Phototaxis	n	48	56	47	61
	%	0.46	0.54	0.44	0.56
	p	0.493		0.211
4 Magnetotaxis	n	56	79	73	69
	%	0.41	0.59	0.51	0.49
	p	0.058		0.674
5 Inclination	n	67	34	50	60
	%	0.66	0.34	0.45	0.55
	p	<0.001 **		0.391
6 Reverse Inclination	n	28	91	65	46
	%	0.24	0.76	0.59	0.41
	p	<0.001 **		0.057
7 Vibration	n	54	48	33	67
	%	0.53	0.47	0.33	0.67
	p	0.488		<0.001 **

shows the results of the choice between cardinal directions. Due to the high number of binomial tests performed on the data, the Benjamini–Hochberg procedure was conducted on the p-values, leaving only p-values below q = 0.01 as still significant. Therefore, while marginally significant results with 0.05 > p > 0.01 might indicate tendencies, they cannot be distinguished from Type 1 errors and should, therefore, be interpreted with caution.

In Experiment 1, which was conducted without external stimuli, Physarum showed no overall systematic preference for left or right, except for one test showing a tendency (p = 0.04) to turn left (north) when starting toward the east. While non-significant, the tendency to go north is mirrored in mazes starting west. Overall, Experiment 1 showed a significant preference for going north (p = 0.008). This preference is not found in Experiment 2 on the other clonal line, however, which shows no significant preference for any direction.

Experiment 3, concerning phototaxis, revealed a significant movement toward the right side of the maze when starting toward the south. Under weak light, Physarum showed a tendency to move away from the light to the left when starting toward the west (p = 0.014), but interestingly not when starting toward the east (p = 0.678).

Experiment 4, concerning magnetotaxis, shows mixed results for strong and weak magnetic fields. In strong magnetic fields, an overall tendency to turn left is observed for all cardinal directions together (p = 0.024), although the effect is not strong enough to manifest on the level of any single cardinal direction and does not hold up after correcting for a false discovery rate. In weak magnetic fields, non-significant preferences for going left (north) when starting toward west (p = 0.024) and significant preferences for going right (north) when starting toward east were found (p = 0.008), as well as a preference for choosing right (east) when starting from the north (p = 0.004).

In Experiment 5, using inclination, a clear significant preference for spreading toward north can be seen both in the overall cardinal direction choice (p < 0.001) as well as in the mazes starting toward west (p = 0.008), but only marginally significant toward east (p = 0.044). Moving north corresponds to moving downward in this setup. Experiment 6, in which the setup was rotated, shows a similar preference for moving south instead, both in the overall cardinal directions (p < 0.001) as well as in the mazes starting toward west (p < 0.001) and east (p = 0.002), again moving downward. The strong downward movement, regardless of cardinal direction, implies a reaction to gravity rather than Earth’s magnetic field lines. Additionally, a non-significant preference for turning left when starting toward the south was observed (p = 0.046), together with an overall non-significant preference for turning left (p = 0.040).

In Experiment 7, testing for the effects of vibration, it was found that when given the chance, Physarum moved significantly more often west toward the vibration (p < 0.001). Furthermore, in all mazes, Physarum showed a significant preference to turn left rather than right, regardless of the relative position of the source of the vibration, showing true lateralized movement under this condition.

Overall, we found significant evidence for laterality in Physarum when confronted with external stimuli, but mostly not without it.

4. Discussion

While behavioral lateralization is a well-studied phenomenon in multicellular animals, it has rarely been studied in unicellular organisms. The first study on lateralized movement in the true slime mold Physarum polycephalum by Dimonte et al. showed a preference for moving right in 75% of the tested plasmodia [8]. To further investigate this phenomenon, we aimed to replicate this experiment and studied the effects of genotype and external stimuli such as light, magnetic fields, gravity, and vibration. Our results suggest that the origin of lateralized movement in Physarum is far more complex than previously thought. While no lateralization was found without stimulus, we showed that side preferences in T-mazes and even true lateralized movement can be induced by various external stimuli.

4.1. Experiments 1 and 2: Lateralized Movement and the Influence of the Genome

Experiment 1 and Experiment 2, which were aimed at replicating the results of Dimonte et al. [8], led to very different results. In contrast to Dimonte et al. [8], neither of the two clonal lines we tested showed intrinsic lateralization under stimulus-free conditions. A minor directional bias appeared in one clonal line (toward the north), but this was absent in the other, highlighting either chance results or potential behavioral divergence across genotypes. Physarum polycephalum is found in a wide geographical range spanning multiple continents [22] and reproduces sexually and, therefore, features a wide genetic variety between populations. While it has not yet been studied in Physarum, high genetic variability, even within populations, has been found in the related species Didymium difforme [20]. Behavioral differences between populations or clonal lines in Physarum have yet to be studied in detail, but the comparison between our data and the results of Dimonte et al. [8] suggest that differences among genotypes might influence plasmodial movement preferences. Regarding lateralized behavior, it would mean that the cellular mechanisms producing lateral preferences in movement are far from universal in Physarum. It is possible that lateralization in Physarum might, therefore, not arise from the same preserved mechanisms found in many organisms but rather represent different genetically encoded foraging strategies. In line with this hypothesis, Dussutour et al. report different, stable foraging strategies between individual plasmodia [23].

4.2. Experiment 3: Phototaxis

In Experiment 3, the influence of light gradients on directional movement was examined. While some statistically significant preferences were observed—such as a rightward turn when starting south and a leftward turn away from light when starting west under weaker light—no coherent pattern emerged across orientations or conditions. The overall lack of systematic effects suggests that light, at least under the conditions used, does not reliably induce lateralized movement. These results do not indicate a systematic reaction to light and, given the relatively small sample sizes, might be due to chance observations. It must be mentioned that to create a gradient, the light had to be mounted to the side of the mazes and, therefore, reached the slime molds at an angle of 55° for the mazes closer to the light and 40° in those farther away. While the angle of light should play no major role in the phototaxis of Physarum [24], it created shadows inside the mazes and, therefore, may have allowed for light evasion of the plasmodia even without displaying clear photophobic movement. In comparison to Experiment 1, it must also be noted that the observed preference for moving north was not observed here. Another explanation might be that the positive magnetotaxis and negative phototaxis of Physarum may have been in conflict and, therefore, caused this indecisive behavior.

4.3. Experiment 4: Magnetotaxis

The results of this experiment show a great influence of magnetic fields depending on field strength. In the weak magnetic field from north to south, given the choice, Physarum preferred to move north. This orientation within the weak magnetic field is in accordance with the findings of Shirakawa et al. [25]. Physarum starting toward the north also significantly more often chose to turn right when forced to decide; however, no similar effect could be observed in Physarum starting toward the south. Contrastingly, the strong magnetic field gave rise to an overall lateralized movement in Physarum, with a preference for moving left in all mazes, regardless of cardinal direction. Interestingly, this mirrors the results of Dimonte et al. [8] despite them not having applied magnetic fields. Therefore, while our Physarum clone line did not show initial lateralized movement toward the left, this could be induced by strong magnetic fields. Shirakawa et al. report that the direction of magnetotaxis reverses depending on the strength of the magnetic field [19]. In our experiment, the induced lateralization seems to reverse correspondingly. Furthermore, in weak magnetic fields, the magnetotaxis seems to be stronger than the lateralization, and it is possible that under both conditions, magnetotaxis, and induced lateralization are influenced and/or overlap with each other if, as the results suggest, induced lateralization is dependent on magnetic field strength. Future studies testing in even stronger magnetic fields could lead to the observation of more pronounced lateralized movement. The mechanism behind this induced lateralization is yet to be investigated. One hypothetical explanation might lie in the actomyosin complex that forms the internal structures of the plasmodial cytoskeleton. The Ca²⁺-activated rhythmic disorganization and reorganization induce changes in the rigidity of the cell, therefore inducing oscillation and allowing for movement through the extension of pseudopodia [26]. A recent study shows, albeit in rat muscle cells, that weak magnetic fields alter the actin polymerization and, therefore, cytoskeleton dynamics [27]. Since the actomyosin complex dynamics is the mechanism behind the plasmodial movement, it is possible to hypothesize that altering the molecular dynamics alters the resulting movement as well, resulting in atypically lateralized movement. This, however, cannot be inferred from the results of this experiment. Whether such molecular perturbations are sufficient to produce consistent behavioral asymmetries in Physarum remains to be tested.

4.4. Experiments 5 and 6: Inclination

The experiments concerning inclination were performed to investigate the effect of truly aligning the mazes with Earth’s magnetic field. In this regard, in Experiment 5, a very clear influence of the stimulus on the behavior was observed, as plasmodia chose north (down) over south (up) when given the opportunity while otherwise not showing lateralized movement. Experiment 6, however, makes it clear that this effect is probably not due to magnetotaxis but rather to gravitaxis: after rotating the setup, Physarum preferred moving south (down) instead of north (up). Therefore, while Experiment 1 suggests a slight preference for moving north, it seems probable that gravity has a stronger influence on Physarum taxis choices.

4.5. Experiment 7: Vibration

The results of Experiment 7 reveal two previously unknown behaviors in Physarum. First, it shows that plasmodia, given the choice to move toward or away from the source of vibration, significantly prefers to move toward it. This behavior has not been reported before, and therefore, no current theories to explain it exist in the literature. A possible explanation could be linked to the fact that Physarum polycephalum feeds on fungal spores and decaying fungal fruiting bodies [28], and fungal fruiting bodies emit weak vibrations as they grow. Therefore, moving toward vibration might be part of Physarum’s natural foraging strategies. For another explanation, it must be considered that the behavior of Physarum is mainly a result of its complex internal network oscillations, created by contraction and relaxation of the cell membrane, thereby moving cytoplasm throughout the cell and guiding movement through expansion [26]. This complex oscillatory system reacts to various stimuli relevant to the organism, such as sources of food or harmful stimuli, but may also be disrupted by external forces or even by the stiffness of the substrate the cell is moving on [29]. While more research is needed to determine if this behavior is ecologically plausible or the effect of disruption of Physarum’s internal oscillatory system, the fact that those organisms starting toward west and east, with no choice to move toward or away from the source of vibration, both showed lateralized movement toward left regardless of the source of vibration, leads us to assume that vibration alters the oscillatory state and, therefore, behavior as a whole.

4.6. General Discussion

Our results show that to discuss lateralization in Physarum, we need to distinguish between general lateralization of a population, stimulus-dependent directional choices, and stimulus-induced general lateralized movement. In our experiments, stimulus-dependent directional choices were the most prevalent behavior of the plasmodia. Only through the T-maze complex design, where plasmodia start towards four different cardinal directions, were we able to determine that some movement occurred relative to the position of a stimulus. Therefore, we argue that if all plasmodia always start in the same direction, the reaction to an unknown stimulus might produce seemingly lateralized movement in T-mazes. Furthermore, plasmodia seem very sensitive to some stimuli, and it is quite possible that not all stimuli influencing movement have been discovered. While magnetotaxis in Physarum has been described for the first time in 2012 [19], to the best of our knowledge, the active movement toward a source of vibration has not yet been described in the literature. We show that one clonal line seems more sensitive to magnetic fields than the other, implying individual differences in sensitivity and possibly even preferences among different genotypes. Thus, further studies of Physarum movement, both regarding lateralization and other movements such as taxis and preferences, should include robust controls, even down to the genotype, to produce reliable results. Taking the new findings of the present study, it is possible that the observations of Dimonte et al. [8] might have been the result of unknown stimuli(us). We argue that Physarum, as a formless organism, does not show general lateralization in a stimulus-free environment. While the cytoskeletal mechanisms for lateralization in other eukaryotic organisms might be evolutionarily preserved among many taxa, they do not necessarily have to be preserved in Physarum as well. The cytoskeletal structure of the Amoebozoa is different from that of other eukaryotes, with an unexpected complexity of microtubule structures, allowing for their unique modes of locomotion [30]. Therefore, we assume that Physarum and possibly other Amoebozoa did not preserve these mechanisms and, therefore, do not show lateralized movement, or if they do, possibly not for the reasons other eukaryotes do.

This, in turn, raises the question of induced lateralization. Both strong magnetic fields and vibration were able to induce a preference for moving left in Physarum, seemingly regardless of the position of the stimulus. Furthermore, while Dimonte et al. [8] describe zig-zag movements and corresponding structures in the tubuli of their plasmodia, in our experiments, these were mostly observed under strong magnetic fields and vibrations. In all our other experiments, we observed only longer, straight tubuli. Both stimuli generally do not occur in the natural environment and, for that reason, may exert a marked influence on the internal systems of Physarum, especially the Ca²⁺-driven rhythmic disorganization and reorganization of actomyosin complexes as the mechanism behind oscillation and movement. The zig-zag pattern might be the result of induced instability in the reorganization or a disturbance of the rhythm due to the magnetic field influencing the flow of calcium ions. Lastly, induced laterality may be the result of stress and general disorientation of the plasmodium due to stimulus intensity, making it more likely to retreat into behavioral heuristics such as behavioral lateralization and disoriented movement. Overall, the mechanism behind induced lateralization remains to be further explored.

4.7. Outlook

The results presented show two phenomena not previously addressed in research: induced lateralized movement and an attraction to vibrational stimuli. Both should be examined in further experiments. Physarum should be tested under a variety of intensities and frequencies of vibration to assess at which point vibration starts to influence Physarum movement and if attraction toward vibration is separate from induced lateralized movement. These should be tested both in the forced-choice paradigm of T-mazes as well as on agar plates, allowing for unrestricted movement to observe whether and under what circumstances movement irregularities occur. Furthermore, substrate type and density should be additional variables in these experiments, as it has been shown that agar density can impact Physarum movement tendencies [29]. It remains to be seen whether attraction to vibration is an ecologically relevant behavior or, rather, whether vibration inhibits physiological processes that guide movement.

5. Conclusions

Lateralization in Physarum polycephalum is more complex than previously thought. While Physarum shows no lateralized movement in the absence of stimuli, some stimuli are capable of influencing choice preferences in T-mazes, while others seem capable of inducing true lateralization. The mechanisms behind the latter are yet unclear, so further study is needed both on the stability of these effects and possible molecular mechanisms behind them. The lack of lateralized movement in the absence of stimulation is a positive factor both for modeling purposes and behavioral experiments, as they do not need to take into account general lateralization. But, for further behavioral studies, caution is needed: once again, the sensitivity to outside stimuli has been shown, and further studies need to consider the possible influence on plasmodial movement. Finally, the newly discovered attraction to vibrational stimuli deserves further scrutiny to uncover possible ecological or physiological mechanisms behind it. Overall, the subject of lateralized movement and the general behavior of Physarum polycephalum remains a fascinating subject with many phenomena remaining that need to be tested.