Observations of linear aggregation behavior in rotifers (Brachionus calyciflorus).

Linear aggregation is present in some animals, such as the coordinated movement of ants and the migration of caterpillars and spinylobsters, but none has been reported on rotifers. The rotifers were collected and clone cultured in the laboratory at 25 ± 1°C, under natural light (light intensity ~130 lx, L:D = 14:10). The culture medium(pH = 7.3) was formulated as described by Suga et al., and rotifers were fed on the micro algae Scenedesmus obliquus grown in HB-4 medium to the exponential growth stage. When density was high (150 individuals ml-1), the behavior of rotifers was observed using a stereo microscope (Motic ES-18TZLED). In this paper, linear aggregation in Brachionus calyciflorus was found for the first time, and experiments were carried out to verify the correlation between linear aggregation and culture density of B. calyciflorus. With the increase of density, the number of aggregations increase, the number of individuals in the aggregation increased, and the maintenance time of the aggregation was also increased. Therefore, we speculate that the formation of aggregates is related to density and may be a behavioral signal of density increase, which may transmit information between density increase and formation of dormant eggs.

Introduction

Little research has been done on rotifer behavior. Mating behavior of the rotifer was described: Epiphanes senta [1], Brachionus plicatilis [2] and Brachionus calyciflorus [3]. Mating behavior of B. plicatilis [2] and B. calyciflorus [3] was described as five phases: encounter, circling, coronal localization, sperm transfer, and dissociation. The oviposition behavior of the littoral rotifer Euchlanis dilatata was investigated by Elizabeth in 1989 [4]. Researchers investigated the effect of toxic substances treatments on swimming speed of Brachionus calyciflorus: bromate [5] and TiO2 nanoparticle [6]. Kim et al. investigated the effects of five external factors on movement in females of Brachionus plicatilis sensu stricto: food limitations, temperature, salinity, predator, and un-ionized ammonia [7]. The effect of UV-B radiation on the feeding behavior of the rotifer Brachionus plicatilis was displayed by Feng et al [8]. But there is no report on the behavior of linear aggregation in rotifer.

Social animals employ certain types of organized behaviors to achieve tasks that are difficult to accomplish as individuals. For example, ants are known to form a chain [9] to carry heavy objects. According to the Daily Mail [10], some 136 caterpillars formed an orderly queue and wriggled head-to-tail across a road in single file. Similar group behaviors are also exhibited spiny lobsters, which form a chain in mass migrations, this behavior may serve a defensive function [11]. However, we are not aware of any other reports that rotifers, an asocial animal, form daisy chain-like associations. We report that rotifers form daisy chain-like associations, providing evidence that this organized social behavior is present among invertebrates living in the zooplankton.

Materials and methods

Freshwater rotifers, Brachionus calyciflorus were collected from a small pond on the campus of Hainan Medical University, Longhua District, Haikou City, Hainan Province in China (Longitude:110.19, Latitude:19.58). The rotifers were collected and clone cultured in the laboratory at 25 ± 1°C, under natural light (light intensity ~130 lx, L:D = 14:10). The culture medium (pH = 7.3) was formulated as described by Suga et al [12]. Rotifers were fed on the microalgae Scenedesmus obliquus grown in HB-4 medium [13] to the exponential growth stage. When density was high (150 individuals ml-1), the behavior of rotifers were observed using a stereo microscope (Motic ES-18TZLED).

Under the microscope, the rotifers, B. calyciflorus formed daisy chain-like aggregations of two to six individuals, most of which consisted of two or three individuals (Fig 1). The linear aggregation pattern was showed in Fig 2. The daisy chain forms frequently moved quickly in one direction. Notably, only the last rotifer in the line had eggs (dormant or mictic).

Fig 1

The phenomenon of linear aggregation observed by microscope.

Fig 2

The linear aggregation pattern of B. calyciflorus.

We conducted two experiments to examine the formation of linear aggregations of rotifers. In the first experiment, B. calyciflorus were observed at different densities (from 10 to 400 individuals 4ml-1) (Table 1). The individuals used in the experiments were selected at random and placed into 4 ml of culture medium to observe the number of aggregations and the number of individuals in each aggregation. The results are recorded in Table 1. In the second experiment, B. calyciflorus multiples of two (to a total of 32 individuals) were placed into 1 ml of culture medium and the number of aggregations formed, and the time over which they persisted, was recorded for ten minutes each density experiment (Table 2). At last, we add the high density B. angularis into the low density B. calyciflorus, the B. calyciflorus also form daisy chain-like aggregations of two individuals.

Table 1

Experimental results of correlation between density and linear aggregation.

Number of B. calyciflorus (Individuals/4 ml)		10	50	100	200	300	400
Number of multiple connectomes	dimer	0	2	3	9	8	9
	triplet	0	0	1	1	2	3
	quadruple	0	0	0	0	1	2
	quintuple	0	0	0	0	0	1

Table 2

Number and duration of dimer occurrences in ten minutes.

Test order	Individuals/1 ml	Number of dimer occurrences	Duration of the dimer(seconds)
1	2	0
2	4	0
3	6	0
4	8	0
5	10	0
6	12	0
7	14	0
8	16	0
9	18	1	23
10	20	1	25
11	22	1	142
12	24	5	16	19	5	3	3
13	26	2	7	5
14	28	3	288	70	46
15	30	6	7	49	6	6	5	17
16*	32	8	42	151	21	183	29	24	481	95

* Triplets occurrence for 7 seconds.

Results and discussion

The results indicate that the formation of linear aggregations increases with an increase in the density of rotifers. The first linear aggregation formed at a density of 50 individuals 4 ml-1 in the first experiment, and at 16 individuals ml-1 in the second experiment. The first linear aggregation of three individuals occurred at a density of 32 individuals ml-1 in the second experiment. The longest duration of a linear aggregation of two individuals was 481 seconds in the second experiment at a density of 32 individuals ml-1. In general, linear aggregations were more stable at higher densities.

Linear aggregations of fossil arthropods were found by Hou et al. They believe that the formation of the stable linear structure found for this species is unique among arthropods, whether fossil or living [14]. Vannier et al. also found linear aggregations of fossil arthropods in Morocco in 2019 [15]. However linear aggregations do not seem to be common among living invertebrates. We are the first to record linear aggregations of living rotifers in culture.

Studies have shown that bacteria perceive and respond to changes in population density through a communication system between cells, which is called quorum sensing. In this system, small molecular hormones called ‘autoinducers’ are produced, released, detected and responded to by bacteria. With bacterial population growth, the concentration of autoinducers released by bacteria also increases. When the concentration of these signal molecules reaches a critical value, bacteria can detect their presence, then amplify the signal, resulting in the expression of target genes [16]. Whiteley et al reviewed progress in and promise of bacterial quorum sensing research. Bacterial communicate with each other and to coordinate their activities by quorum sensing signals. The researches of genetics, genomics, biochemistry, and signal diversity of QS were also summarized [17]. The phenomenon of linear aggregations in B. calyciflorus occurs at high population densities. This may be a behavior signal, which stimulates the expression of specific genes in B. calyciflorus. This could result in the appearance of males and mictic females, leading to mating and the production of dormant eggs. Further research is needed to understand the molecular mechanism that stimulates the production of dormant eggs when B. calyciflorus forms linear aggregations.

Further research is also required to understand whether linear aggregations will occur in other rotifer species, or for other cryptic species of B. calyciflorus, as density increases during the culturing process. At present, this phenomenon has not been observed in cultures of B. angularis, B. caudatus, B. diversicornis, B. forficula, B. quadridentatus, B. urceolaris, B. rubens, B. falcatus, or B. patulus in our laboratory. In high-density cultures of cryptic species of B. calyciflorus, the formation of linear aggregations could also provide a warning that density is too high, providing visual guidance for the large-scale culture of this species.

Linear aggregation may give predators the illusion of size and avoid prey, just like spiny lobsters [11]. Whether the linear aggregation of B. calyciflorus is related to predation pressure needs to be confirmed by relevant experiments. Whether the linear aggregation of B. calyciflorus causes the aggregated rotifers to save motor energy and use more energy for reproductive activities remains to be verified by further experiments.

B. calyciflorus and B. havanaensis exhibited costly and effective defences after induction by predator (Asplanchna brightwelli) infochemicals [18]. Is linear aggregation of B. calyciflorus also caused by some infochemicals? And does linear aggregation of B. calyciflorus product new infochemicals? The answers need more deeply researches.

Video of linear aggregates of B. calyciflorus at high density.

(MP4)

Click here for additional data file.

30 Mar 2021

PONE-D-20-32010

Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)

PLOS ONE

Dear Dr. Cheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that this manuscritpt has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 14 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ram Kumar, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Author

I have been able to get your manuscript reviewed by two reviewers. Both the reviewers suggest minor revision before your manuscript can be accepted for publication. I am also of the opinion that the manuscript need revision, and consequences of linear aggregation behaviour in view of escape from copepod and fish predation and fitness of egg carrying zooplankton like Brachionid rotifers should be elaborated. Formatting should be done carefully and recent published information should also be discussed and elaborated.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Funding Section of your manuscript:

"This study was supported by the Hainan Medical College Talent Introduction

Research start-up project (XRC190013); The National Natural Science Foundation of

China (Grant No. 31860035). The funders provides financial support."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript"

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

**********

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: The manuscript entitled "Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)" discusses linear aggregation in Brachionus calyciflorus upon increase of density and such behavioral pattern among asocial animal like rotifers is an interesting finding. The manuscript may be accepted with minor corrections. The authors need to correct various formatting issues, spelling and grammatical mistakes e.g. " mL-1" etc. Similarly, references also need formatting and

more latest references should be included such as Nature, 2017, 551,313 on bacterial quorum sensing etc.

Reviewer #2: The manuscript entitled "Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)" discusses previously undocumented, linear aggregation in rotifer Brachionus calyciflorus in response to increased culture density. The novel findings are well documented and presented. The manuscript may be accepted with few minor corrections. The author shall review and correct various formatting, grammatical and spelling errors and arrange the references correctly.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 May 2021

Dear Editor,

Thank you for carefully reviewing our manuscript previously titled “Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)” for possible publication in the PLOS ONE. We are grateful to you and your reviewers for their constructive critique. We have revised the manuscript, highlighting our revisions in red and have attached point-by-point responses detailing how we have revised the manuscript in response to the reviewers' comments below.

We added reviewe of rotifer behaviors, and cited some latest references, and corrected various formatting issues, spelling and grammatical mistakes e.g. and arranged the references correctly.

Thank you for your consideration and further review of our manuscript. Please do not hesitate to contact us with any further questions or recommendations.

Yours Sincerely,

shuanghuaicheng2018@hainmc.edu.cn (SC); yanshijiao@hainmc.edu.cn (SY); yunxiazhang@hainmc.edu.cn (YZ); lvchuanzhu677@126.com (CL).

Reviewer Comments:

Reviewer #1: The manuscript entitled "Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)" discusses linear aggregation in Brachionus calyciflorus upon increase of density and such behavioral pattern among asocial animal like rotifers is an interesting finding. The manuscript may be accepted with minor corrections. The authors need to correct various formatting issues, spelling and grammatical mistakes e.g. " mL-1" etc. Similarly, references also need formatting and more latest references should be included such as Nature, 2017, 551,313 on bacterial quorum sensing etc.

Response:

Thank you for your reviewing of our article and giving suggestions. We have modified it according to your suggestions and submitted the revised manuscript. Including the spelling of words, grammar mistakes, the format of the references and so on. And add some latest references including such as Nature, 2017, 551,313 on bacterial quorum sensing etc.

Reviewer #2: The manuscript entitled "Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)" discusses previously undocumented, linear aggregation in rotifer Brachionus calyciflorus in response to increased culture density. The novel findings are well documented and presented. The manuscript may be accepted with few minor corrections. The author shall review and correct various formatting, grammatical and spelling errors and arrange the references correctly.

Response:

Thank you for your reviewing of our article and giving suggestions. We have modified it according to your suggestions and submitted the revised manuscript. Including the spelling of words, grammar mistakes, the format of the references and so on.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Aug 2021

Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)

PONE-D-20-32010R1

Dear Dr. Cheng,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arumugam Sundaramanickam, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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.

Reviewer #1: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

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

Reviewer #1: N/A

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

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

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

**********

6. 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: Authors have satisfactorily addressed all the points raised. Manuscript is now acceptable for publication in the Journal

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

10 Aug 2021

PONE-D-20-32010R1

Observations of linear aggregation Behavior in Rotifers (Brachionus calyciflorus)

Dear Dr. Cheng:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Arumugam Sundaramanickam

Academic Editor

PLOS ONE