PONE-D-20-06920

More nest entrances affect collective decision-making and foraging efficiency in ants.

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Reviewers' comments:

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Comments to the Author

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

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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 manuscript, Lehue and Detrain continue their exploration of the role of the nest entrance on ant foraging. Specifically, they ask how having one or two nest entrances affects the recruitment and foraging dynamics of Myrmica rubra colonies foraging on two sucrose feeders of different qualities (0.1 vs 1M. They report that while having two nest entrances causes a larger surge of recruitment to newly discovered food sources, foraging efficiency suffers for multiple reasons. Key amongst them is that foraging is less well-focussed on the high quality feeder. In addition, fewer recruits successfully find the food source within 10 minutes of leaving the nest.

I found this work interesting, well carried out, and generally well written. The methodologies used were appropriate, and I could detect no critical methodological issues. Almost all of the conclusions drawn are well supported by the data (for one exception, see below). In all, this is a good contribution in an understudied area, and I recommend publication with moderate revisions. However, some key literature is not discussed, and there are methodological choices which may limit the generalisability of the results. Moreover, one of the main results (increased recruitment in 2-entrance nests) did not replicate in the authors previous paper, and the explanation for this was not fully convincing. I also have a series of more minor points about the figures, the supplement, and some grammatical suggestions. I hope the extensive comments I have made show how much I value this work.

Introduction

The introduction is rather long and generic, with the whole first page and a half being basically suitable to any collective decision-making paper on ants. One example is lines 51-56. The authors might consider cutting this generic aspect of the introduction down. They will need this for the next point:

Two papers are key to understanding this work. The first is a previous recent paper by the authors (Lehue et al. 2020), and the second is Pinter-Wollman (2015). The previous work needs to be discussed in much more detail – specifically, all of the main results of that experiment need to be listed, and it should be mentioned that the experiments were identical except for the food sources used. Indeed, the discussion might even benefit from a small table listing the main findings of the current paper and this previous paper, so that we can ‘see’ this body of work in one place.

I was surprised that the Pinter-Wollman paper was not mentioned at all, considering its deep parallels to the current work. Specifically, the paper shows that as connectivity increases, so does recruitment speed. This is very similar to what is reported in the current paper. This paper should thus be discussed in some detail both in the introduction and in the discussion.

Ln 33 – information sources

Ln 33-34 – some of these references are not about social insect decision making, but the sentence is only about that.

Ln 91 – kept, not hosted.

Methods

The food sources were rather close to each other. The resultant pheromone trails from the multiple nest entrances thus cross over quite extensively. I wonder, if the angle between the food sources and the nest were larger (at the extreme, if the food sources were at each side of the nest), whether the effects described in this paper would be present at all. This is probably an issue worth raising in the discussion – how generalisable are these results?

A second methodological concern is the size of the nest: these are quite small nests (300 workers, 8cm diameter), and the distance between the two entrances is quite small (c. 3cm, right?). Thus, as the author notes, the ‘activation area’ of the two nest entrances overlap, possibly leading to increased recruitment. However, in a natural nest, which covers more ground and is composed of a tunnel system, multiple nest entrances likely do not overlap in effect. Again, it is important to ask: how realistic and generalisable are the results, given these methods? I would argue that, if the nest were composed of a tunnel system, and the two nest entrances were in two somewhat separate cavities (as likely occurs in nature), very different dynamics might occur. This does not mean that the current experiment is meaningless – far from it – but it does suggest that we don’t have the whole story here (see the Pinter-Wollman paper, for example). I think this issue needs to be raised explicitly. Maybe in the future, this experiment could be repeated with a nest composed of tunnels and cavities…

Ln 198 – define “ants present at a food source” more formally. Touching the food source? Within 1cm of the food source?

Results

Table 1 – while later in the manuscript it is claimed that the amount of sucrose taken in by colonies is given (ln 370-373), this does not seem to be what is reported here. I note, for example, that 26.1 + 0.8 = 26.9 (column 3). This suggests that the 0.1M sucrose has not, in fact, been reduced by a factor of 10 to account for having only 10% as much sucrose as 1M. The key foraging metric here, after all, is not weight of sucrose solution returned, but the weight of sucrose returned. 1 molar sucrose has ten times as much sugar as 0.1M. Please amend this, and rerun the analysis, if necessary. It will only strengthen the results.

A large concern for me was that one of the main results – the increase in forager mobilisation from 1 to 2 nest entrances – was not replicated in Lehue et al. (2020) (discussed in ln 409-411). This does not make much sense to me, and the hypothesis proposed by the authors is unconvincing. They argue, if understood them correctly, that more food sources would mean a higher rate of returning workers, so higher recruitment and activation. Firstly, given the cross-shaped feeders and presumably no queuing at the feeders, this doesn’t sound likely. Secondly, it is not clear to me why have one or two nest entrances should affect the total number of returning foragers, regardless of the number of food sources. One possibility is that, with only one food source or only one entrance, recruitment is downregulated, due more encounters with ants on the path (Czaczkes et al. 2013) and at the food source (Wendt et al. 2020). However, luckily the authors have all the data needed to support or reject their hypothesis in their videos. If they were to count the number of returning foragers (assessable by abdomen distention) within the first 10 minutes, their hypothesis predicts many more returning foragers in the two-entrance configuration. Don’t forget to collect this data blind to treatment, though, as abdomen distention is quite hard to assess in M. rubra! As an aside, examining the raw data from this and the previous paper, I note that that the foraging dynamics of the colonies are very similar for one food source one entrance (previous paper), one food source two entrances (previous paper), and two food sources two entrances (this paper), with the two food sources two entrances standing out, with double the recruitment. This suggests there is something special about this configuration – it might be worth presenting the data from the 2020 paper again here for comparison.

Ln 468 – replace “dispatching of multiple information” with “distributing incoming information”

Ln 498 – replace “to draw” with “for creating”

Supplement - data

I applaud the authors for providing the raw data. All papers should do this. However, the data is presented in a very hard to use format in the supplement. Firstly, all labels are in French – this should be amended. Secondly, the data has not been entered in a ‘tidy’ manner – tidy having a specific definition in data entry (https://vita.had.co.nz/papers/tidy-data.html). In short – every column should be a variable, and every row an observation. Please also provide metadata (information about what each column is). This all may sound terrible and pedantic (I apologise!), but it really is very important, and will facilitate data analysis and visualisation for you in the future.

Figure 2, 3, 4A&B– please add mean connect lines. Indeed, this data is not normally distributed, so you should probably be using medians and quartiles, not means and SD.

Figure 4 A and B – These figures make seeing the main comparison (1 vs 2 nest entrances) very difficult, and are more appropriate for exploring the dynamics, which is a side issue here. Perhaps just present the total mean time for one and two nest entrance? Indeed, just mean proportion ants at feeder (two bars) captures everything we need to know, but a figure similar to figure 8 would also be ok.

Figure 4C – why does this now have connection lines but no error ribbons?

REFERENCES CITED

Czaczkes TJ, Grüter C, Ratnieks FLW (2013) Negative feedback in ants: crowding results in less trail pheromone deposition. J R Soc Interface 10:. https://doi.org/10.1098/rsif.2012.1009

Lehue M, Collignon B, Detrain C Multiple nest entrances alter foraging and information transfer in ants. Royal Society Open Science 7:191330. https://doi.org/10.1098/rsos.191330

Pinter-Wollman N (2015) Nest architecture shapes the collective behaviour of harvester ants. Biology Letters 11:20150695. https://doi.org/10.1098/rsbl.2015.0695

Wendt S, Kleinhölting N, Czaczkes TJ (2020) Negative feedback: Ants choose unoccupied over occupied food sources and lay more pheromone to them. Journal of The Royal Society Interface. https://doi.org/10.1098/rsif.2019.0661

Reviewer #2: I found this to be a carefully conducted study. It shows that ants are less capable of choosing a richer food source over a weaker one, when the ants have more than one exit hole through which they can leave their nest. Two related things concerned me about the study, which may well be answerable.

1. The short time period during which it seems that the colonies were examined. Do ants improve if they are allowed to forage freely for a few days with two entrances before being tested? You say they were allowed ad lib food for a time with two entrances but without specifying the details. Would be nice to find out if ants do improve over time, by, for instance, coming to use just one of the exits.

2. Could the lack of adaptation to the problems that you set in part be a consequence of abnormal conditions in the artificial nests during testing? E.g. they haven't time to sort themselves out?

Line by line commentary

Line 78. Somewhere around here you need a bit of natural history about nest entrances. There are obvious examples of nests with one entrance (e.g. Cataglyphis fortis). Are there any with several exit holes (Formica rufa ?) and in such cases do individual ants always use the same hole to enter or exit by same hole or do they have other methods of successful recruitment?

Line 100. Be nice to know rough numbers of ants in wild colonies to compare with the 300 in your experimental ones.

Line 118. Do you have evidence that 48 hours is long enough for ants to be acclimated? Might they organise themselves to cope with 2 entrances if given a longer period?

Line 140. Did ants have the chance to explore the arena during this ad lib period or was the food adjacent to the nest and exploration prevented by a barrier? Relevant to general comment at the start of these comments.

Line 240. Was there any correlation between asymmetry of entrance use and proportion of visits to the high concentration feeder?

Line 272. Do you have proportions of how many ants in the one and two nest conditions returned after feeding and how many returned empty stomached?

Line 282 Fig. 4 would be helpful if you label in legend and fig the three conditions (legend) and arrows (fig) as 'a', 'b' or 'c'.

Line 291. I'm curious whether you videoed the foraging and if so whether you can say anything about trail use in the two entrance conditions.

Line 293 'as the 0.1' not 'than the 0.1'

Line 299 Again question about asymmetry of entrance use and proportion of ants reaching 1M sucrose. P = 0.053 doesn't mean no preference but just a weak preference.

Line 305. Don't like 'repartiton' prefer something like 'how ants distributed themselves between..'

Line 333. So there is a correlation between asymmetry of entrance use and food preference. Be helpful to a reader to mention it briefly earlier - around line 240.

Line 348. suggest: 'drank about twice as much from the 1M than from the .1M feeder'

Line 394.409 'twice as many nestmates'

Line 419 -425. I'm uncertain about the argument made here. You need more detail to be convincing that in this experiment the outflow of foragers depends highly on encounters between returning ants and potential foragers. Are there sufficient returning foragers to generate the timing of maximum outflow in Fig 2 when there are two exits? And do returning ants return to their exit hole or are they indiscriminate? Have you compared outflow from one and two hole nests when holes are opened after a period of being shut and there is no food to find. Does the total peak then differ between the two nests? Para needs a conclusion of what the answer might be.

Line 430 'at the same time'

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Reviewer #1: Yes: Tomer J. Czaczkes

Reviewer #2: No

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Foraging through multiple nest holes: an impediment to collective decision-making in ants

PONE-D-20-06920R1

Dear Dr. Detrain,

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

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

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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

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

Reviewer #1: Yes

**********

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

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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

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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: The authors did a good job addressing all the comments. This is a good piece of work, and I look forward to seeing it published.

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Reviewer #1: Yes: Tomer J. Czaczkes