Spatial localization ability of planarians identified through a light maze paradigm

Renzhi Qian; Yuan Yan; Yu Pei; Yixuan Zhang; Yuanwei Chi; Yuxuan Chen; Kun Hao; Zhen Xu; Guang Yang; Zilun Shao; Yuhao Wang; Xinran Li; Chenxu Lu; Xuan Zhang; Kehan Chen; Wenqiang Zhang; Baoqing Wang; Zhengxin Ying; Kaiyuan Huang

doi:10.1371/journal.pone.0288118

. 2023 Jul 19;18(7):e0288118. doi: 10.1371/journal.pone.0288118

Spatial localization ability of planarians identified through a light maze paradigm

Renzhi Qian ^1,^#, Yuan Yan ^1,^#, Yu Pei ^1,^#, Yixuan Zhang ¹, Yuanwei Chi ¹, Yuxuan Chen ¹, Kun Hao ¹, Zhen Xu ¹, Guang Yang ¹, Zilun Shao ¹, Yuhao Wang ¹, Xinran Li ¹, Chenxu Lu ¹, Xuan Zhang ¹, Kehan Chen ², Wenqiang Zhang ², Baoqing Wang ¹, Zhengxin Ying ¹, Kaiyuan Huang ^1,^*

Editor: Elias Garcia-Pelegrin³

PMCID: PMC10355441 PMID: 37467232

Abstract

Spatial localization ability is crucial for free-living animals to fit the environment. As shown by previous studies, planarians can be conditioned to discriminate directions. However, due to their simplicity and primitiveness, they had never been considered to have true spatial localization ability to retrieve locations of objects and places in the environment. Here, we introduce a light maze training paradigm to demonstrate that a planarian worm can navigate to a former recognized place from the start point, even if the worm is transferred into a newly produced maze. This finding identifies the spatial localization ability of planarians for the first time, which provides clues for the evolution of spatial learning. Since the planarians have a primitive brain with simple structures, this paradigm can also provide a simplified model for a detailed investigation of spatial learning.

Introduction

Spatial localization, the ability to learn and remember locations and routes, is crucial to survival for most animals. The ability can be used to forage for food, return to sites of storage or safety, and avoid sites of danger, thus helping animals survive and fit the environment. Thus this ability is considered to have evolved in almost all species [1].

Planarians are free-living flatworms that usually live and stick under rocks, debris and water plants in streams, ponds, and springs [2]. They are considered one of the first class of animals to have a centralized brain structure, and they had evolved various sensory abilities, including sensitivity to light [3], temperature [4], water currents [5], chemical gradients [6], vibration [7], magnetic fields [8] and electric fields [9]. For freely living in the environment, it is reasonable to hypothesize that planarians have evolved spatial localization ability. The study of planarian behavior mainly focused on simple classical conditioning and proved that planarians can learn simple associative tasks. Moreover, planarians are concluded to have abilities to discriminate directions in a Y or T maze [10]. With such ability, they can turn their moving directions in response to a certain stimulus. However, due to the primitiveness and simplicity of planarians, they are not considered to have the ability of true, complex spatial localization ability [11], which is used to localize a certain place in a familiar environment.

To prove the hypothesis that planarians have evolved spatial localization ability, we designed a light maze paradigm inspired by the Morris water maze paradigm [12]. The Morris water maze, designed by Morris in 1981, is widely used to evaluate the rats’ spatial localization ability through distal localization (when the goal object is invisible, inaudible and cannot be detected by smell) rather than proximal localization (when the goal object is visible, audible or detectable by smell) [12]. The Morris water maze uses water as the aversive stimuli to prompt the rats to swim to a platform. Since planarians have innate behavior to avoid light [3], similar to the Morris water maze, we chose light as the aversive stimuli to prompt planarians to move to the dark chamber in the maze. The planarians’ primitive eyes can only sense the light rather than imaging; thus, they cannot visualize the dark chamber. To avoid any chemical cues that a worm might leave in the dark chamber, in the test phase, worms were transferred into newly manufactured mazes from their original mazes. Thus, the information that a worm use to navigate to the dark chamber can only be distal. In the light maze, the distal cues provided to the worm could be such as the topography of the maze, the light intensity gradient, or the geomagnetic field which the goal object did not provide direct information to the worm. The proximal cues could be such as chemicals from the goal object which can be directly smelled by the worms. In the text, we use the word “escape” to show that the worms solved the maze and successfully entered the dark chamber. We also use the escape latency to measure the worms’ performance, which is most commonly used in the Morris water maze, defined as the time for the animal to find the dark chamber and escape the maze. Our light maze paradigm is also inspired by the Barnes maze, which uses open spaces as aversion stimuli to prompt rodents to seek shelter.

Here we demonstrated that planarians were able to use distal information to localize a dark chamber in the light maze. To evaluate the learning effect of the worms, we measured the escape latencies and drew typical routes of worms which showed learning effects. These are the first findings suggesting that planarian worms have spatial localization abilities, indicating that planarians might have higher cognition ability that had never been considered. As a primitive forerunner of more evolved animals, the study of planarian cognition can be evolutionarily instructive to the study of more evolved animals. Also, since the planarians’ nervous system has simple structures, it can provide a model for spatial learning studies.

Materials and methods

Planarians

A laboratory strain of D. japonica, originating from wild collected D. japonica (identified by cytochrome c oxidase subunit 1 gene) from the Cherry-Valley in Beijing Botanical Garden, Haidian district, Beijing, China in 2019. Worms were maintained in Montjuic Water(Simulated components of fresh water from Montjuic) in the dark and fed with chicken liver twice a week on Monday night and Thursday night. The procedure in planarian rearing used protocol from M. Shane Merryman et al. 2018 for reference [13]. The length of planarians used in the experiment varied from 1 cm to 1.5 cm.

Experimental setup

To investigate the spatial localization ability of the planarians, we designed a light maze. It is a square maze with a dark chamber near one corner (Fig 1A). The maze consists of a square pool with a side length of 65 mm and a depth of 6mm, the corners of the maze are rounded to make it easier for planarians to crawl. A square dark room with inner side length of 14 mm and outer side length of 24 mm is located in one of the quadrants as the destination of the maze. The starting position is located in the corner of the opposite quadrant of the dark chamber. The dark chamber has only one entrance. 15 mL Montjuic water is added to the maze before starting a trial.

Fig 1 — A. The conceptual drawing of the light maze. B. The experimental setup of the light maze paradigm. C. The blueprint of the light maze. The upper right shows the lateral view of section A. Length unit: mm. D. Light intensity distribution of the maze. The light intensity distribution is measured according to vertical distance from Wall a. Light intensity variance along Wall a is less than 5 Lux, which can be neglected. E. Diagram of the training procedure.

The mazes were 3D-printed. The material used for 3D printing is photosensitive resin and PLA (Polylactic acid), the main body of the mazes was printed in white using photosensitive resin, and the dark chamber lid was printed in black using PLA (The dark chamber lid can be detached from the main body of maze). In the SLA printing method, a photocurable and photosensitive polymeric material is placed on a surface apparatus and is subsequently opened to a UV light to cure the resin to form a initial layer. When initial layers cure, the repetition of this process fabricates the 3D printing product [9]. The toxicity released by the stereolithography (STL) printing materials (photosensitive resin in this experiment) is fatal to worms, which causes the worms to die and disintegrate in hours, even in the mazes handled by ultraviolet to have reduced toxicity. Although the fused deposition modeling (FDM) materials like PLA is non-toxic, it is of lower printing precision, and its water leaking problem may cause worms to escape from the maze while training. We found that a biocompatible encapsulation material called Parylene to totally block the toxicity from the STL printed parts, using which can make worms safely live in the maze without health problems for over 2 weeks. The inner side of the mazes was coated with Parylene film (thickness of 12 μm) to block the toxicity from the resin [14]. The structure and parameters of the maze are shown in (Fig 1C).

During training, the mazes were put in a line on a black acrylic board to avoid light reflections, and each maze was filled with 15 mL of Montjuic Water. A LED lighting tube was set above the entrance of the dark chamber (Fig 1B) to make the entrance lighted and avoid direct guidance to the entrance by the light. The length of the LED tube was 1.2 m, and the power of the LED tube was 13 W. The distance between the bottom of the LED lighting tube to the acrylic board was 125 mm. The LED lighting tube was set right above the dark chamber. Light intensity distribution of the maze is shown in Fig 1D. Worms were carefully manipulated through a smooth woolen brush.

Training procedure

We totally used 36 worms (2 worms were excluded due to self-fission in the training process) and they were divided into 6 groups. We used 6 mazes to train each group of worms in the first 3 trials. In trial 4, all mazes used to train any of the worms were changed to newly manufactured (36 new mazes). The training procedure started at 8:00 p.m. and ended at 9:00 p.m. each day for a total of 6 trials for each worm in 6 days. 1 day before the training, worms were taken out from the home well to a petri dish. Diagram of the training procedure is shown in Fig 1E. To control worms’ satiety, they were fed at 10:00 a.m. on day 1 and day 4 in a 12-well plate, each worm in a single well. Before training, worms were taken to a 12-well plate, then the water in the mazes is changed and the mazes were set up as described in the last section. The worm is put 5 mm away from wall a and wall b (the starting point might be a little different in each trial because the worm need to sink to the bottom). The room temperature during training was controlled at 20±1°C. After the training, the worms were not taken out, and the maze along with the acrylic board was put in an incubator to control the environment temperature.

Statistical analysis

36 worms were involved in training and 2 of them were excluded due to self-fission. However, possibly because the worms were gradually getting habituated to the light and the environment, about one third of the worms in each trial quickly got to rest and stop moving in each trial except trial 1. Hence, our data excluded worms that rest for more than 20 mins in 40 mins in each trial. So, from trial 1 to trial 6, n = 34, 24, 24, 24, 23, 22. The latency of other worms which did not enter the dark chamber (either rested or kept on moving) were counted as 40 mins. Also, worms that went into the dark chamber but got out within 30 mins did not count as a successful escape.

For statistical analysis, because the escape latencies in trial 1 and trial 4 were not normally distributed (Kolmogorov–Smirnov test), we used the nonparametric Mann–Whitney U-test to evaluate statistical significance of latencies in trial 1 and trial 4. One way ANOVA was applied to determine whether there is a statistical significance between escape latencies of trial 2, 3, 4, 5 and 6. One way ANOVA and subsequent Turkey’s multiple comparison test was applied to determine whether worm speed in trial one is significantly higher than other trials. All data were analyzed using PRISM (GraphPad Prism 9.0.0(121)). Routes are drawn by hand with Photoleap (2.13).

Ethics statement

The overall study and animal experiments of this manuscript conformed to the guidelines and regulatory standards of the Institutional Animal Care and Use Committee of China Agricultural University and approved by the Institutional Animal Care and Use Committee of China Agricultural University.

Results

We tested for recall of the location of the dark chamber of the worms in trial 4 when the mazes were changed to newly manufactured. In trial 4, because the mazes were new, worms cannot use local cues such as chemicals for navigation. The latency result shows that worms in trial 4 displayed a significantly shorter time to escape compared with trial 1 (two-tailed U-test, P<0.01; Fig 2A). To test whether this behavior is stable after changing the maze, we continued trial 5 and trial 6. Comparing escape latencies in trial 4 to trial 2, 3, 5 and 6, there is no significant difference, which means that changing to newly manufactured maze did not affect worm’s performance in the maze, showing that the worms might not use chemical cues in this case (One way ANOVA, P>0.1; Fig 2A). We also counted the worms that underwent successful escapes. From trial 3 to trial 6, there were 10–12 worms that successfully escaped the maze (Fig 2C). However, from trial 2 to trial 6, a worm might successfully escape the maze in some trials but not in other trials. From trial 2 to trial 6, the number of worms that successfully escaped 0, 1, 2, 3, 4 and 5 times is 3, 2, 17, 8, 3 and 3, and the number of worms that consecutively could not escape the maze for 2, 3, 4, and 5 times is 12, 8, 1 and 3.

Fig 2 — A. Latency of each trial. Data are the average±SEM of worm’s latency for worms moved for more than 20 mins in 40 mins. From trial 1 to 6, n = 34, 24, 24, 24, 23, 22. B. Worm speed in each trial. C. Worms successfully escaped in each trial. C-G. Representative routes of learned worms. C, E show routes of trial 1. D, F show routes of trial 4. C and D are routes of 1 worm and E, F are routes of 1 worm. The green spots show the start point and the red spots show the end point.

To exclude the effect of worm speed on latency, we counted the worm speed of a total moving distance of 241 mm (the perimeter of the maze) for worms which moved for more than 241 mm, otherwise, we used its actual moving distance to count its speed. The result shows that worm speed in trial 1 are significantly higher than other trials (One way ANOVA, P<0.001; Fig 2B), which means the worm speed did not shorten the latencies from trial 2 to trial 6.

To further understand the worms’ learning effect, we showed representative routes of trial 1 and trial 4 of 2 worms which successfully escaped the maze in trial 4 (Fig 2C–2F). In trial 4, the mazes are newly manufactured, and no chemicals or mucus can affect the navigation of the worms. As the worms did not know the location of the dark chamber prior to the training, the routes of trial 1 were mainly distributed on the walls of the maze. In trial 4, the worms that showed learning effects quickly secede from the wall of the maze and find the entrance of the dark chamber, which showed significantly shorter routes than routes in trial 1, demonstrating that worms can use distal information to navigate to their destinations.

Discussion

In our findings, we designed a light maze to demonstrate that planarians were able to use distal information to localize a dark chamber in the light maze. After training, the worms can use shorter time or shorter distances to navigate to the dark chamber. Below, I’ll discuss details and issues in this paradigm.

From the 1950s to the 1960s, numerous studies had been done to understand planarian learning and behavior [15]. The most commonly used procedure is a classical conditioning protocol to make worms associate a light stimulus with an electrical stimulus [16]. Other studies showed that worms can learn more complicated tasks such as discriminating directions in a Y or T maze [10, 17]. However, due to the manual practical difficulties and the control of variables, a large number of research results failed to be reproduced in lots of cases [15]. Consequently, this field became largely abandoned, and even the ability of planarians to form long-term memory was questioned [18].

Some classical conditioning paradigms of the planarians can be attributed to pseudoconditioning or sensitization instead of true learning and memory encoded by the brain [19]. Since spatial learning is a more complex behavior that requires the integration of different neural circuits in higher animals [20], spatial learning might also be a great point to investigate the memory and learning of planarians, and can be regarded as true learning and memory.

The control of variables and manipulations of the experimenters can also largely affect the worms’ behavior. In previous studies, light intensity [21], water temperature [22], water existence [10], time of day [22], time of year [23], chemical components of water [23], chemical components of food [24], worm’s appetite level [25], slime trails [26], worm fatigue state [23], magnetic fields [27], training conditions and manipulation of the experimenter [23] were considered to affect the worms’ behavior. Our light maze paradigm was designed to decrease the number of variables as much as possible to make the experiment easy to reproduce. In our paradigm, the light intensity, water components, water temperature, appetite level and training time of day are strictly controlled.

The fabrication of mazes using 3D printing techniques is a great advantage in this research. Designing such a number of mazes of different shapes requests a much higher cost than 3D printing techniques. With the assistance of the 3D printing techniques, further investigation of the worm’s spatial learning will be much more convenient.

The results demonstrated that worms showed learning effects in each trial. However, a worm might successfully escape the maze in some trials but not in other trials, which makes it a little hard to judge the learning effect of the worm. We believe that this phenomenon might be caused by multiple factors including the habituation phenomenon. I When the worms habituate to the environment and light stimulation, it might ignore the light and choose to rest under the light. Or this is because the navigation ability of planarians might be primitive, and cannot accurately solve every task. So, many worms cannot solve the task in some trials.

Although we endeavored to exclude proximal cues that worms might use to navigate, we still do not understand which kind of distal cues the worms might have utilized. We speculate that the worms used the light intensity gradient combined with the geometric information of the maze to navigate to the dark chamber. The worms might learn to get to brighter place to find the dark chamber. Also, the worms can learn to secede the wall to find the dark chamber. To further investigate this question, we think that the position of the dark chamber can be changed to test whether learned worms can still get into the dark chamber. Also, the electric and magnetic gradients from the led, earth and/or other lab equipment could also be used as distal cues for worms to navigate.

In conclusion, we identified the spatial localization ability of planarians by presenting a paradigm using the light maze we designed. The paradigm can also be a new tool for the analysis of spatial learning in planarians. In this work, planarian worms were found to use distal cues instead of proximal cues to navigate to the dark chamber. Since planarians are now the most primitive animal found to have spatial learning ability, identifying the spatial localization ability of them might provide insights into the evolution of spatial learning.

Supporting information

S1 Data

(XLSX)

Click here for additional data file.^{(30.7KB, xlsx)}

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0288118.r001

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Elias Garcia-Pelegrin

22 Dec 2022

PONE-D-22-32791Spatial Localization Ability of Planarians Identified Through a Light Maze ParadigmPLOS ONE

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, the authors develop a light maze and investigate the spatial localization ability of planarians. They found that planarians succeeded in navigating themselves to a learnt position, suggesting that planarians could learn and remember locations and routes. This paper can be published in a modified form in the future. However, I cannot recommend publication in its present state. Before the paper is considered for publication, there are questions that must be answered:

Major concerns:

Is it correct that successful worms follow the same trajectory as the previous successful trials? Why not calculating the trace accuracy using the data? In fact, you show only two figures regarding the data analysis. Maybe too little analysis? Linked to this, is there a difference regarding the speed of worms between the first trial and the other trials? I wonder the speed of worms is affected by the learning accuracy.

I could not find any statistical explanations regarding the data in Results section. Linked to this, why not adding some statistical analysis to results shown in fig 2B? Are there any statistical differences regarding the ratio of worms reached criterion between trial 1 and the other trials?

I wonder what kind of spatial information worms rely on. What happens if some geometry arrangement in the light maze is modulated after several trials? Also, you may as well consider the rearrangement of the position of the dark chamber after several trials. Could you expand discussion regarding this?

Minor comments:

Training procedures: The training procedures is also written in the first paragraph of Results section. I recommend you that you should move it to Method section. In addition, I found that you said in the “Training Procedure” of Method section that 24 worms were used in the training. However, in Result section, you said that 35 worms were totally used. Which is correct?

Fig1B: Can you make fig1B a little bit larger? In addition, the two boxes on the far left appear to be different in color from the others. Why?

fig2C-F: Although you present some trajectories, I would like to see some movies.

Reviewer #2: The paper suggests a new light-maze system for studying spatial orientation in Planarians.

The idea and the system are interesting and has merits. The paper itself however have some serious flows. The first and most important is that the entire statistics section is completely missing.

There is indeed a part titled "statistics", but it contains no relevant information whatsoever. The results parts follows the same theme – there is a paragraph titled "results" but in actuality it is just an extension of the "Methods" part. Also, because the paper contains no statistical tests, there are no real results reported anywhere in it.

These two issues alone should disqualify the paper, to my opinion.

However, because of the somewhat novel and interesting system – I think it is worth publishing after some major revisions. Mainly: (a) adding the full statistics, (b) rewriting the "Results" and "Methods", and (c) measuring light levels on the maze surface (there is no need to re-run the entire experiment – only arrange the set up as it was and measure light levels across the maze, maybe at the beginning, middle and just above the dark solution patch).

There are also various other issues in the paper which I will list below.

Introduction.

Line 15: Please elaborate more or at least give an example regarding the differences between the "ability to discriminate directions" and "complex localization ability".

Line 15-24: This part deals with the Morris water maze and the light-based system the authors offer. The authors' suggestion and design are solid, but they are in fact a combination of the Morris water maze and the Barnes light maze. I therefore feel like the latter should be at least mentioned here.

Line 24-25: ("The planarians’ primitive eyes can only sense the light rather than imaging; thus, they cannot visualize the dark chamber". I am not sure 'visualize' is the correct word here and would have chosen a slightly different phrasing.

Line 26: The text jumps to introduce "trial 4" without describing first the experimental design or mentioning previous trials or their purpose. I would (a) consider moving this entire paragraph to "Methods" (b) change "trail 4" to "test phase" or something similar along these lines.

Line 31: "This finding proves that the planarian worms have spatial localization ability, which is not yet discovered". One cannot claim to report something which is not yet discovered. I would rather write something like "these are the first finding suggesting that planarian worms have spatial localization abilities".

Lines 34-35: ("Also, since the planarians’ nervous system has simple structures, it can

provide a model for spatial learning studies."). This sentence is true and it makes sense. However, it remains at the statement level if there is no follow up, elaboration or a suggestion for further research. Similar statements repeats through the paper, but still without any concrete suggestions.

Materials and Methods.

Line 3-4: First, past form is preferable here (worms were instead of worms are). Second, I feel like a short explanation of what exactly "Montjuic Water" are is due here. I for once don't know this term and reference 13 is locked behind a pay-wall. Also, if this is indeed a standard procedure in planarian rearing I would like to see some references to this method: for example "Worms were maintained in Montjuic Water in the dark (like in XX et-al. 2019, YY & ZZ 2022) etc…."

Finally, food amounts and preparation methods are missing: How much chicken liver? In what form? Was it whole? Crushed? Minced? Was it mixed in the water? Was it removed the next day?

Experimental Setup.

Line 2: Why are the maze dimensions not mentioned in the text? Please describe the maze and mention them before you are referring me to the Figures (although they should be mentioned there too)

Line 3: Extra 'r' in 'were' (here the paragraph is back to past tense, which is good). Also, why was photosensitive resin used for the maze? It is probably nothing and I am sure the authors have a good explanation and they thought this out. But since this paper deal with light mazes and it effect on small creatures I would have like to see this issue somehow been addressed. It would be reassuring to the reader to explain why it is not a problem.

Line 6: ("The inner side of the mazes is coated…"). We are back to present tense, please don't jump from tense to tense, at least not within the same paragraph.

Line 10-15: This discusses the lightning of the maze. In light maze experiments it is common to measure and report the illumination level (in Lux), rather than state the specifications of the lightning apparatus. If specifications already appear, I would also like to see the makers/brand of the Led tube reported – This could help a lot in recreating the experiment.

In light of the the results mentioned in the Discussion - It would be a good idea to re-set the experiment (no animals or water needed) and to measure and report the light level at least at the beginning and end points of each maze. Otherwise the authors cannot claim the worms relied only on distal cues to solve the maze.

Training procedure:

This entire part is very unclear and inconsistent. I feel it should be re-written completely and therefore there are no line numbers stated here but only general remarks.

Some unclear and troubling points:

1. this sections starts with: "We used 6 mazes to train 24 worms in the first 3 trials".

Were the worms trained individually or in groups of 4? Did water or mazes were switched between training runs? (Something like that was mentioned before - but it is not mentioned here).

2. Next the text states: "total of 6 trials in 6 days" - isn't this come up to a total of 34 trials (6 treatments X 4 trials equals 24 trials). What is the reason for this discrepancy?

3. "To control worms’ satiety, they were fed at 10:00 a.m. on day 1 and day 4"

When were the worm fed? On Sunday and Thursday as mentioned before? A day before training? Were Sunday and Thursday both were a day before training? This is very unclear.

Also - Why is important to control satiety when there is no food reward in the maze and the initiative to solving it is light avoidance? If it is only for standardization, say so. If there is another reason - mention it.

4. "Then the water in the mazes is changed (add to 15 mL) and the mazes were set up as described above." – Well, (a) nothing is described above, (b) were the water changed or were water added to fill a certain minimum? Also, why?

5. While re-writing this section, it might be useful to add an explaining diagram describing trials, times, number of worms and the entire experiment process.

Statistical Analysis:

This is the entire statistical analysis section: "Statistical Analysis All data were analyzed using PRISM (GraphPad Prism 9.0.0(121)). Routes are drawn by hand with Photoleap (2.13)."

Need I say more? Please add Statistics…

Also, please explain the process of drawing the routes, as I understand it - Photoleap is a drawing software, not a movement tracking one.

Results:

This part should also be re-written. In reality it is not a result section but a continuation of the "Methods" part. This is probably somewhat due to the fact that since the statistical tests are missing, there is nothing to report.

This part should contain only the results of each important step of the experiments (including the test-names, the correct statistic for each test, the number of repetitions/iterations and the numbers of animals used in each such tests).

This entire sections should be re-written and moved to the methods, so here too – I will address some issues in general. Some of these are important and alarming.

1. The numbers of worms and trials here are different from what is described earlier (in the methods section).

Why were 35 worms here and either 24 or 34 in previous part?

The executions times are also different from what is mentioned in the methods (although this might be the result of an am/pm typo). If this is the results section – how come a different number of worms was used? If this is actually a continuation of the Methods section – why is it under Results?

2. Worms' satiety is mentioned again here – the question remains, why is it important in the framework of a light maze?

3. "During the training process, only **partial worms** showed learning and got to the dark chamber **fast** in each trial." (a) I assume "partial worms" means "some worms"? Please rephrase. (b) What does fast mean here? Is it important? (explain why), is it trivial? (omit it).

Is "fast" on a scale? Is it only binary fail/success? More importantly - are Fast/Slow definitions meaningful within your experimental design? If so, they need to be defined, measured and reported accordingly.

Lines 3-4: "In each training trial, a planarian was put in the start point (opposite the dark chamber corner)". Please add this start point to the figures. Do this not only in the hand drawn route figures (in which the starting point slightly differs - but also in the relevant places in Figure 1.

Line 14: "If a worm stopped moving outside the dark chamber for more than 20 mins, its data from that single trial is excluded" – What does that mean? I think it means that worms that didn't move for more than 20 min. were omitted from the statistics. But I can't be sure. Also, because you don't report the statistics, it is hard to understand if or how meaningful it is. Please explain/rephrase and include all relevant information (including the number of cases where this happened). Also elaborate about your "learning criterion" while at is. When were 40min. used and where did you use 20 min.?

Line 16-17: "From trial 3 to trial 6, there were 10-12 worms that showed learning effects in each trial (Fig2.B). This part starts to resemble "results". Please phrase it more clearly. Also you have to mention (clearly) somewhere what is your exact "learning criterion" (Is it "worm got to the shade in under 20min.? something else? Don't leave me guessing) and add it to the graph/figure. Also add the statistical test results (is 7 in trial 2really statistically different from 10 in trial 4? Should I guess here too?).

Lines 17-18: "Comparing trial 4 to trial 2, 3, 5 and 6, there is no significant difference, showing that the worms might not use chemical cues in this case (Fig2.A)". Here you are jumping to a different graph showing something different than the one two lines ago, yet you are still speaking about trial numbers only (rather than training/test runs or other more informative terms).

This is very confusing and should be corrected. More important, you are claiming there are significant differences here but do not bother to back this statement up: What test did you use? What were the results? Where are the statistical values?

Discussion:

Lines 3-4: Why are these lines relevant here? It has nothing to do with either the lines preceding or following it.

Lines 6-8: examples needed here - also, are you sure this belongs in the discussion and not in the introduction?

Lines 10-14: You are comparing between planarians and mice here. While It is true that one animal have a simpler brain than the other, just stating this fact does not make it a "great investigation point" – please back this statement up by either explaining why is it such a great opportunity and/or at least suggest a theoretical way to test this.

Lines 14-16: the lines state "As in our paradigm, we applied the light maze paradigm to identify the spatial localization ability of the planarians, which can be regarded as true learning and memory."

First, the words "As in our paradigm" does not connect to the previous sentence. Second, please explain what do you mean by "true learning and memory" (are there false learning and memory within your paradigm? Do you mean to differ it from other kind of learning? If so – which? And why?). Otherwise just omit this part.

Line 24-25: "The results demonstrated that…"

1. Normally this is how you should have started the discussion.

Here however there are no results. What appears under "Results" is actually a continuation of the methods section and the Statistics and Results sections are completely missing.

2. "A worm might show learning effects in some trials but not in other trials." – While this can happen, you can't just wave this aside without addressing the issue. You need to state why do you think it happened, how often, what does it mean, and if it is a problem or not. Although some explanations follow, they are confused, not sufficient and does not address some of the issues I just mentioned. For example, you cannot just say "the planarians navigation system is primitive and that is why they can solve some tasks and not others". Please explain at least what tasks you are referring to (not to mention what sets apart the unsolved ones). Also, from the initial phrasing it follows that worms also differed in learning results within the same task. Please rephrase to address these issues.

Lines 34-35: The authors suggests the "worms used the light intensity gradient combined with the geometric information of the maze to navigate to the dark chamber." – This is another reason why it is important to measure the light gradient across the experiment arena. As already mentioned above – this is something I like to see done before submitting this paper again. Also, how exactly was the geometric of the maze used by the worms?

Finally, as was mentioned by the authors earlier in the article – the worms can detect also vibration, and magnetic and electric fields, how did they account for, or controlled these variables? (i.e - no talking in the experiment chamber, isolating electric devices etc...)

Line 36: "we identified the spatial localization ability of planarians by presenting a new paradigm using the light maze we designed." – No you did not, you cannot make this claim without presenting the statistics supporting it. Also, as mentioned above - this paradigm is far from being new. If anything, you applied an existing paradigm to a new model animal.

Lines 38-39: "In this work, planarian worms were found to use distal cues instead of proximal cues to navigate to the dark chamber. I am sorry, but I have to ask again – can you really say that? The authors themselves just stated light gradient as a possible explanation for their findings, and a few other factors (electric and magnetic gradients from the led, earth and/or other lab equipment) could also have influence the results. Please measure the light gradient and also rephrase this part.

Lines 40-41: "Identifying the spatial localization ability of such a primitive invertebrate might provide insights into the evolution of spatial learning". Once again, this stays at the statement level. Even if this is true, please elaborate how this provide insights, what kind of insights do you think it will provide, what do you suggest as a direction for future research, where do you think this should go next – things like that..

Figures: Figure 2C,D,E,F – These suppose to represent the learned routes of the worms. However, as the text indicates they were drawn by hand using Photoleap. Since Photoleap is not a movement tracking software but rather a 'freehand' drawing program, I would like to see more information regarding the methods by-which the worms movement was translated from the test arena to the final figures. Also, why is the start point in each of them slightly different?

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Reviewer #2: No

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PLoS One. 2023 Jul 19;18(7):e0288118. doi: 10.1371/journal.pone.0288118.r002

Author response to Decision Letter 0

31 Jan 2023

Major concerns:

We thank the reviewer for pointing out these issues. However, after trying some software, we could not find an effective tracing software that can accurately trace the worms. Because when the worm climbs on the sidewall of the maze, the software loses the target. This might also happen when a worm wriggle and causes its body to change shape. Therefore, we chose to draw some routes to prove that the worms could somehow get to the dark chamber. Also, by roughly estimating the worm’s trace, we did not find a general rule for how the worms got to the dark chamber. Even so, our video can be provided and used for analysis by other researchers. We also counted the speed of the worms and found that worms are getting slower after the first trial, which leads to a respectively longer latency for worm to get to the dark chamber. Data analysis of the speed of the worms is now added in the result part.

We thank the reviewer for pointing out this issue. We apologize for omitting statistical analysis. Statistical analysis of the original data is now added to the result part.

We thank the reviewer for this suggestion. Due to technical restrictions on our route tracing method, we are still working on this question. The discussion part is expanded regarding to the suggestion.

Minor comments:

We thank the reviewer for pointing out this issue. The number of worms is now corrected.

Fig1B: Can you make fig1B a little bit larger? In addition, the two boxes on the far left appear to be different in color from the others. Why?

We thank the reviewer for pointing out this issue. Fig1B is now larger. The photosensitive resin material tends to change its color to a little yellow when exposed under light. However, since planarians do not have visual abilities, we don’t think that this factor might affect the result.

fig2C-F: Although you present some trajectories, I would like to see some movies.

We thank the reviewer for this suggestion. The movie can be provided.

Reviewer #2: The paper suggests a new light-maze system for studying spatial orientation in Planarians.

The idea and the system are interesting and has merits. The paper itself however have some serious flows. The first and most important is that the entire statistics section is completely missing.

These two issues alone should disqualify the paper, to my opinion.

There are also various other issues in the paper which I will list below.

We thank the reviewer for pointing out these issues. The statistics were added and the relevant parts were rewritten as suggested.

Introduction.

Line 15: Please elaborate more or at least give an example regarding the differences between the “ability to discriminate directions” and “complex localization ability”.

We thank the reviewer for the suggestion. The relevant part is now elaborated more as suggested.

Line 15-24: This part deals with the Morris water maze and the light-based system the authors offer. The authors’ suggestion and design are solid, but they are in fact a combination of the Morris water maze and the Barnes light maze. I therefore feel like the latter should be at least mentioned here.

We thank the reviewer for the suggestion. The Barnes light maze is now mentioned in the text.

Line 24-25: (“The planarians’ primitive eyes can only sense the light rather than imaging; thus, they cannot visualize the dark chamber”. I am not sure ‘visualize’ is the correct word here and would have chosen a slightly different phrasing.

We thank the reviewer for pointing out this issue. The sentence is now rephrased as “The dark chamber is not visible to the worms.”

Line 26: The text jumps to introduce “trial 4” without describing first the experimental design or mentioning previous trials or their purpose. I would (a) consider moving this entire paragraph to “Methods” (b) change “trail 4” to “test phase” or something similar along these lines.

We thank the reviewer for pointing out this issue. The phrase “trail 4” is changed to “test phase”.

Line 31: “This finding proves that the planarian worms have spatial localization ability, which is not yet discovered”. One cannot claim to report something which is not yet discovered. I would rather write something like “these are the first finding suggesting that planarian worms have spatial localization abilities”.

We thank the reviewer for pointing out this issue. The sentence is rephrased as suggested.

Lines 34-35: (“Also, since the planarians’ nervous system has simple structures, it can

provide a model for spatial learning studies.”). This sentence is true and it makes sense. However, it remains at the statement level if there is no follow up, elaboration or a suggestion for further research. Similar statements repeats through the paper, but still without any concrete suggestions.

We agree. In fact, currently, there is little research about planarian behavior, nor did we find any neural circuit studies of planarians, possibly due to the difficulty of gene editing in planarians. Therefore, we could not currently give concrete suggestions and we hope that further studies can solve this problem to help establish this field.

Materials and Methods.

Line 3-4: First, past form is preferable here (worms were instead of worms are). Second, I feel like a short explanation of what exactly “Montjuic Water” are is due here. I for once don’t know this term and reference 13 is locked behind a pay-wall. Also, if this is indeed a standard procedure in planarian rearing I would like to see some references to this method: for example “Worms were maintained in Montjuic Water in the dark (like in XX et-al. 2019, YY & ZZ 2022) etc….”

Finally, food amounts and preparation methods are missing: How much chicken liver? In what form? Was it whole? Crushed? Minced? Was it mixed in the water? Was it removed the next day?

We thank the reviewer for pointing out this issue. We actually used protocol from M. Shane Merryman et al. 2018 for reference and this case is now illustrated in this part.

Experimental Setup.

Line 2: Why are the maze dimensions not mentioned in the text? Please describe the maze and mention them before you are referring me to the Figures (although they should be mentioned there too)

We thank the reviewer for pointing out this issue. The maze dimensions are now added to the text.

Line 3: Extra ‘r’ in ‘were’ (here the paragraph is back to past tense, which is good). Also, why was photosensitive resin used for the maze? It is probably nothing and I am sure the authors have a good explanation and they thought this out. But since this paper deal with light mazes and it effect on small creatures I would have like to see this issue somehow been addressed. It would be reassuring to the reader to explain why it is not a problem.

We thank the reviewer for pointing out this issue. The explanation of this issue is now added to the discussion part.

Line 6: (“The inner side of the mazes is coated…”). We are back to present tense, please don’t jump from tense to tense, at least not within the same paragraph.

We thank the reviewer for pointing out this issue. The tense is now corrected.

In light of the the results mentioned in the Discussion – It would be a good idea to re-set the experiment (no animals or water needed) and to measure and report the light level at least at the beginning and end points of each maze. Otherwise the authors cannot claim the worms relied only on distal cues to solve the maze.

We thank the reviewer for pointing out this issue. The lighting conditions is now added in the method part.

Training procedure:

This entire part is very unclear and inconsistent. I feel it should be re-written completely and therefore there are no line numbers stated here but only general remarks.

Some unclear and troubling points:

We thank the reviewer for pointing out the issues below. The entire part is now rewritten and clearly illustrated the issues below.

3. this sections starts with: “We used 6 mazes to train 24 worms in the first 3 trials”.

Were the worms trained individually or in groups of 4? Did water or mazes were switched between training runs? (Something like that was mentioned before – but it is not mentioned here).

2. Next the text states: “total of 6 trials in 6 days” – isn’t this come up to a total of 34 trials (6 treatments X 4 trials equals 24 trials). What is the reason for this discrepancy?

3. “To control worms’ satiety, they were fed at 10:00 a.m. on day 1 and day 4”

When were the worm fed? On Sunday and Thursday as mentioned before? A day before training? Were Sunday and Thursday both were a day before training? This is very unclear.

Also – Why is important to control satiety when there is no food reward in the maze and the initiative to solving it is light avoidance? If it is only for standardization, say so. If there is another reason – mention it.

Satiety control is for standardization and is illustrated in the discussion part.

4. “Then the water in the mazes is changed (add to 15 mL) and the mazes were set up as described above.” – Well, (a) nothing is described above, (b) were the water changed or were water added to fill a certain minimum? Also, why?

Water is changed. Now, the water amount added to the maze is illustrated in the Experimental Setup part.

5. While re-writing this section, it might be useful to add an explaining diagram describing trials, times, number of worms and the entire experiment process.

A diagram is now added.

Statistical Analysis:

This is the entire statistical analysis section: “Statistical Analysis All data were analyzed using PRISM (GraphPad Prism 9.0.0(121)). Routes are drawn by hand with Photoleap (2.13).”

Need I say more? Please add Statistics…

Also, please explain the process of drawing the routes, as I understand it – Photoleap is a drawing software, not a movement tracking one.

We thank the reviewer for pointing out this issue. We apologize for omitting statistical analysis. Statistical analysis of the original data is now added to the result part.

Results:

This part should also be re-written. In reality it is not a result section but a continuation of the “Methods” part. This is probably somewhat due to the fact that since the statistical tests are missing, there is nothing to report.

This entire sections should be re-written and moved to the methods, so here too – I will address some issues in general. Some of these are important and alarming.

We thank the reviewer for pointing out these issues. The entire section is now rewritten, and problems described below were all solved.

3. The numbers of worms and trials here are different from what is described earlier (in the methods section).

Why were 35 worms here and either 24 or 34 in previous part?

The number of worms is now corrected. The entire section is now rewritten.

2. Worms’ satiety is mentioned again here – the question remains, why is it important in the framework of a light maze?

Satiety control is for standardization and is illustrated in the discussion part.

3. “During the training process, only **partial worms** showed learning and got to the dark chamber **fast** in each trial.” (a) I assume “partial worms” means “some worms”? Please rephrase. (b) What does fast mean here? Is it important? (explain why), is it trivial? (omit it).

Is “fast” on a scale? Is it only binary fail/success? More importantly – are Fast/Slow definitions meaningful within your experimental design? If so, they need to be defined, measured and reported accordingly.

The entire part is now rewritten and the sentences relevant were rephrased.

Lines 3-4: “In each training trial, a planarian was put in the start point (opposite the dark chamber corner)”. Please add this start point to the figures. Do this not only in the hand drawn route figures (in which the starting point slightly differs – but also in the relevant places in Figure 1.

The start point is now illustrated in the text.

Line 14: “If a worm stopped moving outside the dark chamber for more than 20 mins, its data from that single trial is excluded” – What does that mean? I think it means that worms that didn’t move for more than 20 min. were omitted from the statistics. But I can’t be sure. Also, because you don’t report the statistics, it is hard to understand if or how meaningful it is. Please explain/rephrase and include all relevant information (including the number of cases where this happened). Also elaborate about your “learning criterion” while at is. When were 40min. used and where did you use 20 min.?

The relevant part is now rephrased and clearly illustrated.

Line 16-17: “From trial 3 to trial 6, there were 10-12 worms that showed learning effects in each trial (Fig2.B). This part starts to resemble “results”. Please phrase it more clearly. Also you have to mention (clearly) somewhere what is your exact “learning criterion” (Is it “worm got to the shade in under 20min.? something else? Don’t leave me guessing) and add it to the graph/figure. Also add the statistical test results (is 7 in trial 2really statistically different from 10 in trial 4? Should I guess here too?).

The relevant part is now rephrased and clearly illustrated.

Lines 17-18: “Comparing trial 4 to trial 2, 3, 5 and 6, there is no significant difference, showing that the worms might not use chemical cues in this case (Fig2.A)”. Here you are jumping to a different graph showing something different than the one two lines ago, yet you are still speaking about trial numbers only (rather than training/test runs or other more informative terms).

The relevant part is now rephrased and clearly illustrated.

Discussion:

Lines 3-4: Why are these lines relevant here? It has nothing to do with either the lines preceding or following it.

We thank the reviewer for pointing out this issue. The sentence redundant and is now deleted.

Lines 6-8: examples needed here - also, are you sure this belongs in the discussion and not in the introduction?

We thank the reviewer for the suggestion. However, this point is too complicated to explain in this section and is well illustrated in the reference 15. Thus, we decide not to give an example here. And this part is relevant to the next paragraph that we are going to discuss, so we think that this belongs in the discussion part.

We thank the reviewer for the suggestion. However, there is currently zero investigation on behavior and neural circuit of the planarian worm. Thus, could not offer a theoretical way to test this. Anyway, we rephrased the sentences to make it sound reasonable.

We thank the reviewer for pointing out this issue. The sentence is now rephrased.

Line 24-25: "The results demonstrated that…"

1. Normally this is how you should have started the discussion.

Here however there are no results. What appears under "Results" is actually a continuation of the methods section and the Statistics and Results sections are completely missing.

We thank the reviewer for pointing out these issues. The sentences are now rephrased.

We thank the reviewer for pointing out this issue. The word “new” is now deleted.

We thank the reviewer for pointing out this issue. The part is rephrased.

We thank the reviewer for the suggestion. However, there is currently zero investigation on behavior and neural circuit of the planarian worm. Thus, we feel sorry that we could not expand this topic.

We thank the reviewer for pointing out these issues. However, after trying some software, we could not find an effective tracing software that can accurately trace the worms. Because when the worm climbs on the sidewall of the maze, the software loses the target. This might also happen when a worm wriggle and causes its body to change shape. Therefore, we chose to draw some routes to prove that the worms could somehow get to the dark chamber. So, all the routes were drawn freehand through watching the video with Photoleap. The start point in each of them is slightly different because because the worm need to sink to the bottom, and this is when we started drawing.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(30.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0288118.r003

Decision Letter 1

Elias Garcia-Pelegrin

29 Mar 2023

PONE-D-22-32791R1Spatial Localization Ability of Planarians Identified Through a Light Maze ParadigmPLOS ONE

Dear Dr. Huang,

==============================

Thank you for your revised version, please address and reply to the reviewers comments and concerns for this round in order for this manuscript to be reconsidered for publication.

==============================

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Reviewers' comments:

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

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6. Review Comments to the Author

Reviewer #1: The authors have satisfactorily addressed most of my concerns.

I wonder why worm speed in trial 1 are significantly higher than other trials.

You may or may not discuss this.

Reviewer #2: Lines 17-21: These lines suggests claims that the worms can navigate to a formerly recognized place from a start point. For these claims to hold water they need to meet at least one of two conditions: (1) did routes became shorter between runs? (There is no evidence in the text for that), (2) did solution times became shorter between runs (according to the authors answer to the first reviewer, it seems they actually got longer). Unless at least one of this conditions is satisfied, how do we know that the worms do not just randomly swim until they find the save dark patch?

Lines 50-51: I would rephrase these lines or completely omit them. (I think I can guess what the general meaning here is, but it is badly written and confusing and I am not sure how much it contributes to the overall understanding of the design).

Lines 84-85: Here I would have liked to see some reference to the photosensitive resin used. I asked for this matter (why use a photosensitive resin in such an experiment design when photosensitivity is key) to be clarified during the previous revision – and I am asking for this again now.

Line 107: please add the missing space between "changed' and "and".

Lines 117-119: The authors state "Light intensity variance along Wall a is less than 5 Lux, which can be neglected" - Reference is certainly needed here. Also light level should be measured not only from the wall of each maze but also in points between the maze 'entry' and maze 'solution'. From figure 1D it seems quite likely that there is might be some brightness gradient there.

Line 123: This is the first time the term "latency" appears in the text. I do not understand what it means here (or for that matter, when you use it again and again in the following parts of the text). Please define this terms clearly as it seems important to your claims. I would have preferred for it to be discusses or defined in the materials and methods section or in experiment design – but it doesn't really matter as long as you define it clearly the first time it appears in the text.

General remarks for the "statistics" chapter:

This is poorly written and does encourage trust in your results. Please phrase your research questions (preferably at the end of the introduction part), then report the statistical tests you chose to test these questions and why. This part should also include the number of repetitions and of worms in each state (not only of the ones used in the tests, but also the worms disqualified and the reasons for doing so).

Important: Note that you are mentioning specific tests in the "statistics" section and then report the result of *different* tests in the result section.

Lines 142-143: This would be a good place to state the total number (N) of the worm which the authors actually included in the statistics. Also, I do not completely understand what "latency' mean here too – please add an explanation. While we are on this subject – please state clearly not only the N for participating worms, but also for the disqualified ones.

Lines 145-145, 150-151: I would choose a term other than 'escape' and clearly define it. If you choose to remain with 'escape', please add a definition or an explanation for it (does it mean 'solved the light maze?', 'escaped the arena'? something else?). Also fix the extra dot/period in line 150. Note that in line 207 in the discussion (for example) you are using 'escape' in yet another context.

Lines 147-157:

a. these lines states: "To test whether this behavior is stable after changing the maze, we continued trial 5 and trial 6. comparing trial 4 to trial 2, 3, 5 and 6, there is no significant difference, showing that the worms might not use chemical cues in this case (One way ANOVA, P>0.1; Fig.2A)". The text does not specify what was compared to what?! What was the comparison criteria? Speed? Number of solutions? What is behind the statement that this comparison shows "that the worms might not use chemical cues in this case"

b. If there is significant difference between trial 1 and all other trails, indicate it - as you did in 2A. But this is a minor problem. If solving speed declines with trials (as stated in lines 154-157), how can you tell the worms learned? How can you say that there were more "solves" (if that is what 'escapes' means) in advanced trials because of learning and because of random chance (e.g., worms explored the maze for longer times).

c. I ask again, what exactly is "latency" in your experiment system? Also, why use 241mm. in some cases and actual moving distance in others? You are pooling two different data sets together and then running a single test on the new pool? Why?, Please explain this.

Lines 162-163: Please state how many worms we are talking about (this is already the results section and still we don't know how many worms were used for the statistics). What are the learning effects? As mentioned before – if solution times does not diminish and/or solution routes does not shorten, what is the criteria for learning?

Line 169: Please add a space between "40" and "min.

Lines 171-174: please fix this entire paragraph: the worms' routes Illustrations starts with 2D and not with 2C as appears in the text. More important issue here is that for some reason, in the advanced trials illustrations the end point of the worms *are not* under the dark chamber (while ending under it in the first trial). It seems like, at least from the illustrations that in opposition to the authors claims – the worms did not learn.

General remarks for the "Results" chapter:

I would like to see a better written Results part. This should include better definitions of what was measured. (for example: what is 'latency'? what are 'escapes'? how was 'speed' calculated? Why there is no report regarding routes length). Please describe exactly what was measured or tested under each such definition or terms. Do so before or when you are introducing this term for the first time.

You should also state what exactly was compared to what? Did solution times changed between trial 1 and 4? If yes - To what direction? If not – why and what criteria was compared? Why did you choose to compare trial 1 to 4 and not 1 to 6?

I would also like to see the actual number of animals eventually used in each statistical test as this is very unclear in the current text (something is mentioned under fig.2 but it is unclear). The N should appear also in the manuscript main text next to each relevant test. This is especially true when some of the N values mentioned are a bit too small for comfort).

Eventually, I cannot understand why you are describing statistical tests in the 'statistics' section and report results from completely different tests in the results. Please take care of this issue.

Lines 177-184: These lines belongs in the introduction. Please start the discussion with shortly describing your main findings. Then you can move on to explain them in relation to other work in the field or if there is none – their novelty. Either way though - these lines are not relevant here.

Line 192: It seems like the word 'including' is unnecessary here.

Lines 200-211: these lines also do not belong in the discussion. The process of manufacturing 3d mazes, their toxicity and the material used should appear in the introduction and in the method parts. You can of course stress the novelty and advantages of your design and techniques in the discussion – but not to delve into a deep discussion of materials and method. It belongs elsewhere. Also, please notice that in line 207, you are using 'escape' in yet another context.

Lines 212-214: These are the lines the discussion part should have begun with. However:

a. you are using 'escaped' in yet another context here.

b. It is not clear if the results are neither significant or actually support the authors claims.

c. This bring us back to the results section. Please rewrite it.

Lines 214-218: What does this mean for the results? How many times did this happen? This should be stated in the results section. Also, what do the authors mean by "worms cannot accurately solve every task"? Did they control for each worm in successive trials and singled out ones that repeatedly could not solve their light maze? Something else? Since there is no report of the proportions of 'solvers' vs. 'non-solvers' the reader can not evaluate the meaning or strength of this statement.

Lines 230-234: In these lines the authors claims that "In this work, planarian worms were found to use distal cues instead of proximal" and that "planarians are now the most primitive animal found to have spatial learning ability".

In light of the remarks above, I am yet to be convinced: What were the definitions for proximal and distal in this experimental setup? According to what hypothesis or tests the authors reached this results? These should appear in the manuscripts, ideally at the end of the introduction.

General remarks for the discussion:

The discussion also need some rewriting: The entire opening paragraph (lines 177-184) describes the history of the research field and belongs (if at all), in the introduction. The discussion then moves to explain reasons for the results (which should have been briefly summarized in the opening of the discussion). Directly after that the text explains the benefits (and some of the problems) of 3D printing and only on line 212 (35 lines into the discussion) finally some of the results first appear. This is structurally wrong and needs to be fixed.

What worries me more, however, is that what described in the 'results' section not necessarily shows that the worms actually learned between sessions: what is the criteria? Learning speed? Total numbers of worms that found the shelter in each trial? Shortening routes? Did the researchers followed specific worms and saw better performance? The results chapter lack clarity and the researches hypothesis are not mentioned, therefore it is hard to understand what on what bases the authors' states these results.

**********

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Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2023 Jul 19;18(7):e0288118. doi: 10.1371/journal.pone.0288118.r004

Author response to Decision Letter 1

1 May 2023

Reviewer #1: The authors have satisfactorily addressed most of my concerns.

I wonder why worm speeds in trial 1 are significantly higher than other trials.

You may or may not discuss this.

We thank the reviewer for pointing out this issue. When worms encounter an unfamiliar environment, their speed becomes high in order to get familiar with the environment as soon as possible, as worm speed in trial 1 is significantly higher than worm speed in other trial, and worms’ speed gradually decreases trials after trials. Interestingly, when worms are transferred from an old maze to a new maze (maze pattern is the same), their speed slightly increases, as worms’ speed in trial 4 are slightly higher than trial 2, 3, 5 and 6.

Reviewer #2: Lines 17-21: These lines suggest claims that the worms can navigate to a formerly recognized place from a start point. For these claims to hold water they need to meet at least one of two conditions: (1) did routes became shorter between runs? (There is no evidence in the text for that), (2) did solution times became shorter between runs (according to the authors answer to the first reviewer, it seems they actually got longer). Unless at least one of this conditions is satisfied, how do we know that the worms do not just randomly swim until they find the save dark patch?

We thank the reviewer for pointing out this issue. In figure 1.A, we can see that solution times became shorter between runs. In trial 1, the solution time is significantly higher than that of trial 4 (worms were transferred to a new maze). The short solution time means that learned worms can navigate to the dark chamber in a new maze. In Figure 2.D-G, we can see that routes became shorter between trial 1 and trial 4. However, we could not evaluate the exact route distance in each trial due to technical difficulties as demonstrated in our former response. The relevant descriptions are now added to the result part.

We thank the reviewer for pointing out this issue. The sentence is now rephrased.

We thank the reviewer for pointing out this issue. There are mainly two types of 3D printing techniques currently, fused deposition modeling (FDM) and the stereolithography (SLA). The objects produced by FDM are porous and water leaking, which made us not to consider this technique in making the main body of the maze. Also, the SLA 3D printing method which uses photosensitive resin as raw material behaves better in 3D-printing speed and resolution. The photosensitivity of the resin means that during 3D fabrication, the liquid resin cures after exposed to the UV light. After curing, the resin becomes stable, solid and can no longer change its physical properties under any kind of light. Therefore, the photosensitivity of the resin had no relevance to the light in our maze experiment. The reference and the illustration above are now added to the Materials and Methods part of the manuscript.

.Line 107: please add the missing space between "changed' and "and".

We thank the reviewer for pointing out this issue. The missing space is now added.

We thank the reviewer for pointing out this issue. Actually, what we wanted to express is that in a typical maze, the light intensity variance (LIV) along wall b is more than 100 Lux, which is much larger than its LIV along wall a, which is less than 5 Lux. So, if the worms relied on the LIV to navigate, we think that they would rely more on the LIV along wall b than wall a. To avoid misunderstanding, we have now deleted the phrase “which can be neglected”. Based on illustration above, the light level between maze 'entry' and maze 'solution' should be ±5 Lux from our measurement in figure1.D, Based on this, we think it is no need to measure light intensity from everywhere in the maze.

We thank the reviewer for pointing out this issue. The relevant definition is added from line 71-75 in the introduction part.

General remarks for the "statistics" chapter:

Important: Note that you are mentioning specific tests in the "statistics" section and then report the result of *different* tests in the result section.

We thank the reviewer for pointing out this issue. The research question is now added to the end of the introduction part and illustrations of statistical tests are now added to the statistics chapter.

We thank the reviewer for pointing out this issue. The number of worms counted in each trial is now added to the statistics part. The definition of latency is added from line 71-75 in the introduction part.

We thank the reviewer for pointing out this issue. The relevant definition is added from line 71-75 in the introduction part. The extra dot/period in line 150 is now fixed.

Lines 147-157:

We thank the reviewer for pointing out this issue. The detailed explanation of the sentence is now added.

We thank the reviewer for pointing out this issue. There is a logical relationship that if the speed declines with trials, it takes more time for the worms escape the maze, which makes it harder for worms to show their learning effects. However, the decline of the speed did not affect the worms’ escape latencies, so it won’t affect the conclusion that worms learn to escape the maze faster after training.

We thank the reviewer for pointing out this issue. The definition of latency is added from line 71-75 in the introduction part. 241 mm is the perimeter of the maze. For worms which can quickly get into the dark chamber, its moving distance is usually less 241 mm, so we can only use its actual moving distance to count its speed. For other worms who usually climb along the wall of the maze, it is more accurate to use the maze perimeter to count its speed, so we chose 241 mm to count its speed.

We thank the reviewer for pointing out these issues. These issues can be solved by my responses on other issues above: through definition of latency and illustration of effect of worm speed on latencies.

Line 169: Please add a space between "40" and "min.

We thank the reviewer for pointing out this issue. The space is now added.

We thank the reviewer for pointing out this issue. The worms' routes Illustrations are now fixed. The end points were outside the dark chamber is because our camera could not catch the worm routes inside the dark chamber. Anyway, all worms that solved the maze were identified that they were in the dark chamber after the test. And we can provide videos to demonstrate this issue.

General remarks for the "Results" chapter:

You should also state what exactly was compared to what? Did solution times change between trial 1 and 4? If yes - To what direction? If not – why and what criteria was compared? Why did you choose to compare trial 1 to 4 and not 1 to 6?

Trial 4 uses newly manufactured mazes to test the worms, in this case, worms cannot use local cues such as chemicals for navigation, so we compare trial 1 to 4 and not 1 to 6. This illustration locates at the first sentence of the result section.

Eventually, I cannot understand why you are describing statistical tests in the 'statistics' section and report results from completely different tests in the results. Please take care of this issue.

We thank the reviewer for pointing out these issues. We have now added detailed explanation of these issues in the “Statistical Analysis” chapter.

Lines 177-184: These lines belong in the introduction. Please start the discussion with shortly describing your main findings. Then you can move on to explain them in relation to other work in the field or if there is none – their novelty. Either way though - these lines are not relevant here.

We thank the reviewer for pointing out this issue. The discussion part is now started with shortly describing our main findings.

Line 192: It seems like the word 'including' is unnecessary here.

We thank the reviewer for pointing out this issue. The word including is now deleted.

We thank the reviewer for pointing out this issue. Related parts are moved to the method part.

Lines 212-214: These are the lines the discussion part should have begun with. However:

a. you are using 'escaped' in yet another context here.

b. It is not clear if the results are neither significant or actually support the authors claims.

c. This brings us back to the results section. Please rewrite it.

We thank the reviewer for pointing out these issues. We have now added these information in the result part. However, there are only 3 worms in 36 worms that could solve all 5 trials. Other worms can only solve 0-5 trials for some unknown reasons.

Lines 230-234: In these lines the authors claim that "In this work, planarian worms were found to use distal cues instead of proximal" and that "planarians are now the most primitive animal found to have spatial learning ability".

In light of the remarks above, I am yet to be convinced: What were the definitions for proximal and distal in this experimental setup? According to what hypothesis or tests the authors reached these results? These should appear in the manuscripts, ideally at the end of the introduction.

We thank the reviewer for pointing out these issues. However, we did not explore which kind of proximal and distal cues that the worms relied on. Anyway, we listed possible proximal and distal cues in the 3rd paragraph in the introduction part.

General remarks for the discussion:

We thank the reviewer for pointing out these issues. These issues can be solved by my responses on other issues above.

What worries me more, however, is that what described in the 'results' section not necessarily shows that the worms actually learned between sessions: what is the criteria? Learning speed? Total numbers of worms that found the shelter in each trial? Shortening routes? Did the researchers followed specific worms and saw better performance? The results chapter lack clarity and the research hypothesis are not mentioned, therefore it is hard to understand what on what bases the authors' states these results.

Attachment

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Click here for additional data file.^{(28.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0288118.r005

Decision Letter 2

Elias Garcia-Pelegrin

21 Jun 2023

Spatial Localization Ability of Planarians Identified Through a Light Maze Paradigm

PONE-D-22-32791R2

Dear Dr. Huang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Elias Garcia-Pelegrin, Ph.D

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0288118.r006

Acceptance letter

Elias Garcia-Pelegrin

11 Jul 2023

PONE-D-22-32791R2

Spatial localization ability of planarians identified through a light maze paradigm

Dear Dr. Huang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Spatial localization ability of planarians identified through a light maze paradigm

Renzhi Qian

Yuan Yan

Yu Pei

Yixuan Zhang

Yuanwei Chi

Yuxuan Chen

Kun Hao

Zhen Xu

Guang Yang

Zilun Shao

Yuhao Wang

Xinran Li

Chenxu Lu

Xuan Zhang

Kehan Chen

Wenqiang Zhang

Baoqing Wang

Zhengxin Ying

Kaiyuan Huang

Roles

Abstract

Introduction

Materials and methods

Planarians

Experimental setup

Fig 1. Experimental setup and training procedure.

Training procedure

Statistical analysis

Ethics statement

Results

Fig 2. Results of the light maze experiment.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Elias Garcia-Pelegrin

Roles

Author response to Decision Letter 0

Decision Letter 1

Elias Garcia-Pelegrin

Roles

Author response to Decision Letter 1

Decision Letter 2

Elias Garcia-Pelegrin

Roles

Acceptance letter

Elias Garcia-Pelegrin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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