kotahi

Metadata

eLife Assessment

This important study convincingly shows that Vibrio bacteria act as predators of ecologically significant algae that contribute to harmful blooms in the lab and in their natural habitat, and that predation is induced by starvation. The authors suggest a working model that can be the basis for future work on this system. The study will be very impactful to those interested in the diversity of microbial predator-prey interactions and controlling toxic algal bloom.

Reviewer #1 (Public review):

Summary:

Rolland and colleagues investigated the interaction between Vibrio bacteria and Alexandrium algae. The authors found a correlation between the abundance of the two in the Thau Lagoon and observed in the laboratory that Vibrio grows to higher numbers in the presence of the algae than in monoculture. Timelapse imaging of Alexandrium in coculture with Vibrio enabled the authors to observe Vibrio bacteria in proximity to the algae and subsequent algae death. The authors further determine the mechanism of the interaction between the two and point out similarities between the observed phenotypes and predator prey behaviours across organisms.

Strengths:

The study combines field work with mechanistic studies in the laboratory and uses a wide array of techniques ranging from co-cultivation experiments to genetic engineering, microscopy and proteomics. Further, the authors test multiple Vibrio and Alexandria species and claim a wide spread of the observed phenotypes.

Comments on revisions:

I thank the authors for their additional work on the manuscript. My comments were addressed to my satisfaction.

Reviewer #2 (Public review):

Goal summary

The authors sought to (i) demonstrate correlations between the dynamics of the dinoflagellate Alexandrium pacificum and the bacterim Vibrio atlanticus in natural populations, ii) demonstrate the occurrence of predation in laboratory experiments, iii) demonstrate that predation is induced by predator starvation, and iv) test for effects of quorum sensing and iron-uptake genes on the predation process.

Strengths include

- Data indicating correlated dynamics in a natural environment that increase the motivation for study of in vitro interactions
- Experimental design allowing clear inference of predation based on population counts of both prey and predators in addition to microscopy-based evidence
- Supplementation of population-level data with molecular approaches to test hypotheses regarding possible involvement of quorum sensing and iron update in predation

Weaknesses include

- A quantitative analysis of effects of manipulating V. atlanticus density on rates of predation would have been valuable
- Lack of clarity in some of the methodological descriptions

Appraisal

The authors convincingly demonstrate that V. atlanticus can prey on A. pacificum, provide strongly suggestive evidence that such predation is induced by starvation and clearly demonstrate that both iron availability and correspondingly the presence of genes involved in iron uptake strongly influence the efficacy of predation.

Discussion of impact

This paper will interest those interested in the diversity of forms of microbial predation and how microbial predatory behavior responds to environmental fluctuations. It will also interest those investigating bacteria-algae interactions and potential ecological controls of algal blooms. It may also interest researchers of microbial cooperation in light of the suggestion of communication between predator cells.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Weaknesses:

In my view, the presentation of the data is in some cases not ideal. The phrasing of some conclusions (e.g., group-attacks and wolf-pack-hunting by the bacteria) is in my opinion too strong based on the herein provided data.

We agree with your comment and have replaced the terms “Group-attacks” and “wolf-pack-hunting by “attacks” throughout the manuscript.

Reviewer #1 (Recommendations for the authors):

(1) Figure 2AB, please add the name of the statistical test and the number of replicates that the data is based on to the figure legend.

We thank Reviewer#1 for highlighting the need for more detail. We have revised the manuscript accordingly. The captions of figures 2, 3, 4 and S1 were revised to include the name of the statistical test and the number of replicates. Asterisks indicate significant differences in a multiple comparison test (One -way ANOVA with post hoc Tukey test),* P ≤ 0.05, ** P≤0.01, *** P≤ 0.001

(2) Figure 2C is this figure referred to in the text?

We apologize for this oversight. Figure 2C was replaced by new figures 2C and 2D and the old figure 2C is now referenced in the manuscript as Fig 3B1.

(3) Movie 1, could the movie please also be provided as .mp4? I suggest including individual images across time in the main figure so that readers do not rely on opening a supplementary file for this key finding of the study.

In the revised manuscript, all the videos were converted to mp4 format and individual images across time were included in Figure 2C and 2D (Chronological snapshots of one attack) and in figure 3B1 (Chronological snapshots of the complete event), thereby improving the readability of the manuscript.

(4) Figure 3A2 (text l. 355), I am afraid I do not find this figure.

Fig. 3A2 which previously corresponded to Fig. 3B1, correspond now to Fig. 2C and Fig. 2D. This has been corrected in the revised version of the manuscript.

(5) Lines 356ff, I am afraid that I find it hard to follow what the authors refer to as the right cell or the left cell. I suggest either adding labels to the movies or providing individual images across multiple timepoints into the main figure that can be labelled and bring across the point.

Arrows have been added to videos 3–5 to clearly indicate the cells referred to in the text and facilitate tracking across time.

(6) In general, for all the microscopy, on how many cells have these phenomena been observed? What is n=x? Has this been quantified?

We thank the reviewer for pointing this out.

In caption of Fig. 3, the sentence “(A) Percentage of motile A. pacificum ACT03. (B) A. pacificum ACT03 attacked by V. atlanticus LGP32 and (C) A. pacificum ACT03 lysis after 0, 15, 30, 45 and 60 min of interaction. “was replaced by “(A) Cumulative percentage of motile A. pacificum ACT03 cells. (B) Cumulative number of cells attacked by V. atlanticus LGP32 and (C) Cumulative cell lysis after 0, 15, 30, 45 and 60 minutes of interaction.”. In Fig. 3 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was also added.

In Fig. 4 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was added.

In Fig. S1 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was added.

(7) Figure S1A, does this figure show means plus/minus standard deviation? If yes, please add this to the figure legends.

In Fig. S1 caption, the sentence “Error bars represent the standard deviation of the mean of three independent experiments” was added.

How do the authors explain the big variation in the test condition and not in the control?

Regarding the higher variation observed in the test condition compared to the control, this may, on the one hand, reflect biological variability between independent batches of 60-h V. atlanticus cultures used to prepare the supernatants, and, on the other hand, a heterogeneity in the physiological status of independent algal batches (N = 3 ; 2 × 10^4 cells ; see Materials and Methods, Co-culture assay), which may not be perfectly synchronized . In contrast, the control condition consists of A. pacificum cultures incubated in fresh medium without bacterial supernatant, for which algal motility is highly reproducible and thus shows very little variation.

(8) Line 375, "The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Movie 5) and was induced by the old-starved culture supernatant of V. atlanticus LGP32 (Fig. S1)." Is this reference to Figure S1 correct? S1 shows motility, doesn't it? I don't see how this data supports the statement made in this sentence.

We apologize for this unclear message.

"The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Video 5) and was induced by the old-starved culture supernatant of V. atlanticus LGP32 (Fig. S1)." was replaced by "The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Fig. 3C and 3C1).

And “We next tested whether this lytic effect was mediated by thermostable molecule (s) secreted by Vibrio. “was replaced by “We next tested whether this lytic effect was linked to Vibrio culture supernatant and mediated by thermostable molecule (s) secreted by Vibrio.

(9) Line 388ff, "Group attacks were observed on non-degraded A. pacificum ACT03 cells, but not on previously lysed cells." No reference to a figure is provided. I am afraid I don't see the data that this statement is based on.

As it is impossible to show a lack of attack, we just clarified the basis of our experiment.

“To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 60-hour culture of V. atlanticus LGP32, which induced 25% lysis of A. pacificum ACT03 cells. Next, the corresponding V. atlanticus LGP32 cells were added. During exposure, attacks were observed only on undegraded A. pacificum ACT03 cells, but not on previously lysed cells” was replaced by “To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 126-hour culture of V. atlanticus LGP32, which induced lysis of 70% of the A. pacificum ACT03 cells (Figures 3C and 3C1, arrow 2 and video 4). Next, cells of V. atlanticus LGP32 from a 60-hour culture, capable of attacking A. pacificum ACT03 cells (Fig. 3B), were added. For 1 hour of exposure, no attack was observed on the previously lysed algae.”

(10) Figure 4a, Based on the labeling of the figure, in particular the x-axis, it is not fully clear to me what I am looking at.

Figure 4A has been reworked and its legend modified. We hope that this graph is clearer now.

(11) Line 428, did the authors consider complementing the pvuD deletion mutant and testing for gain of function when providing the gene in trans?

We did not investigate pvuD in this study and did not construct a pvuD deletion mutant. We therefore assume that the recommendation refers to pvuB, which was the focus of our work. Unfortunately, we did not perform this experiment. However, several lines of evidence support the implication of PvuB and the vibrioferrin uptake system in this process: (i) the loss of attack behaviour is specific to the mutant in the vibrioferrin uptake pathway and (ii) our expression and proteomic data show a strong induction of vibrioferrin uptake components under starvation and iron-manipulated conditions, which correlate with the attack phenotype.

(12) Use of the term "group attack" in parentheses in the text, but in the section header and title. Is there really sufficient actual data to say that this is a "group attack"? What exactly are the indications for this being a behaviour of a group?

We agree with you. The terms “group attacks” and “wolf-pack hunting” were replaced by the more neutral term “attacks” throughout the manuscript.

(13) Table S1 and S2, those tables give a nice overview. Do the authors provide the raw data based on which they make a claim on "+" and "-" in the individual categories? I would prefer to see the actual data or at least have the possibility to look into this.

In the revised versions of Tables 1 and 2, we have improved the captions and clarified the meaning of each column in order to avoid any ambiguity between the results of this study and the bibliographic information.

Specifically regarding Table 2 :

We do not present any visuals of the interaction between Vibrio and Alexandrium because these species all look alike. Regarding the other algae species tested in interaction with Vibrio, phenomena other than lysis or cell attack have been observed and are the subject of specific laboratory studies.

(14) Line 456 "first study", line 40f "first evidence of a new mechanism". I suggest toning this down a bit and being clearer in the abstract about this being a working model that can be suggested based on individual bits of data.

We thank Reviewer #1 for this helpful suggestion.

In the summary:

“This is the first evidence of a new mechanism that could to be involved in regulating Alexandrium spp. blooms and giving Vibrio a competitive advantage in obtaining nutrients from the environment.” was replaced by “The interaction model we propose here suggests that Vibrio could play a role in regulating the proliferation of Alexandrium spp., giving it a competitive advantage in obtaining nutrients from the environment.”

In the discussion:

Considering predator as a free organism that feeds at the expense of another, this study is the first evidence of the capacity of some Vibrio to develop a predatory strategy against an alga. This behaviour differs from parasitism, because the survival of Vibrio is not exclusively dependent on algae in environment” was replaced by “Consider a predator as a free-living organism that kills its prey and feeds on it, this study provides data suggesting the ability of Vibrios to develop an original predator-like behaviour to kill and feed on algae.”

(15) Line 469 "Overall, these observations show that V. atlanticus LGP32 is able of wolf-pack hunting behaviour." I see the similarities. I feel that the term "show" is a bit too strong here, or I suggest referring to "wolf-pack-like behaviour".

The sentence “Overall, these observations show that V. atlanticus LGP32 is able of wolf-pack hunting attack behaviour” was replaced by “Overall, these observations suggest that V. atlanticus LGP32 can exhibit a predator-like behaviour”

Reviewer #2 (Public review):

As Weaknesses Reviewer #2 include:

(1) A lack of early, clear definitions for several important terms used in the paper, including 'predation', 'coordination' and 'coordinated action', 'group attack', and 'wolf-pack hunting', along with a corresponding lack of criteria for what evidence would warrant use of some of these labels. (For example, does mere simultaneity of attacks of an A. pacificum cell by many V. atlanticus cells constitute "coordination"? Or, as it seems to us, does coordination require some form of signalling between predator cells?)

The term “Coordinate” was replaced by “simultaneous” throughout the manuscript

The terms “Group attack” and “wolf pack hunting” were replaced by “attack” throughout the manuscript

(2) Absence of controls for cell density in the test for starvation effects on predatory behaviour; unclear how the length of incubation affects the density of V. atlanticus cells.

We thank the reviewer for pointing this out.

Cells density experiment was already performed (cf. Fig. 4A).

The sentence. ”All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.“ was added in captions of Fig. 3, Fig. 4 and Fig S1

(3) Lack of clarity in some of the methodological descriptions

The Methodology has been checked and some improvements have been made.

Reviewer #2 (Recommendations for the authors):

(A) Title

(1) Could 'induces' be better than 'promotes'?

We agree with Reviewer #2. The initial title, “Starvation of the bacterium Vibrio atlanticus promotes lightning group-attacks on the dinoflagellate Alexandrium pacificum”, was replaced by “Starvation of the bacterium Vibrio atlanticus induces simultaneous attacks on the dinoflagellate Alexandrium pacificum”.

(B) Abstract

(1) Perhaps define pycosphere in the abstract - many readers might not know this word.

We have revised the abstract to define the term phycosphere and added the sentence “This occurs in the microenvironment surrounding phytoplankton cells, the phycosphere. An interface rich in nutrients and organic molecules exuded by the cell.”

(2) Perhaps "on dinoflagellates".

We thank Reviewer #2 for this suggestion. We have revised the abstract by replacing “on the dinoflagellates species” with “on dinoflagellates”.

(3) Line 33 - The word 'prey' is used without a claim of predation having yet been made; only killing has been claimed so far.

We agree and have replaced the word “prey” by “algae” in the abstract.

(4) Line 34 - It is unclear whether the description refers to the 'attack stage' or to 'wolf-pack attack' in general. The sentence is written in such a way that it seems to refer to 'wolf-pack attack'. However, this would seem to be incorrect, with the description being specific to V. atlanticus.

To avoid this ambiguity, we have removed the sentence “resembles the ‘wolf-pack attack’ strategy” from the abstract.

(5) Line 35 - Should there be a 'consumption phase'?

We agree with the reviewer #2, “degradation” was replaced by “consumption”.

(6) If predation is claimed later in the manuscript (which it is), it should be explicitly claimed in the abstract.

We thank Reviewer #2 for this helpful suggestion.

We have revised the abstract. The sentence “Results showed that Vibrio atlanticus was able to coordinate lightning group attacks then kill the dinoflagellate Alexandrium pacificum ACT03” was replaced by “The results showed that Vibrio atlanticus was capable of attacking and killing the dinoflagellate Alexandrium pacificum ACT03”.

(C) Main text

(1) Line 54 - Perhaps "Among HAB-causing organisms...".

We agree with the reviewer’s suggestion and have revised the wording.

(2) Line 56 - "that, together with..., form the "Alexandrium tamarense" complex".

We agree with the reviewer’s suggestion and have revised the sentence.

(3) Line 57 - What this "complex" is and its significance should be explained.

“Among them, Alexandrium pacificum is a flagellated eukaryotic unicellular organism that together with Alexandrium tamarense and Alexandrium fundyense form the "Alexandrium tamarense" complex (Hadjadji et al., 2020)” was replaced by

(4) Line 58 - What is a Rephy survey?

We clarified this point, “by rephy survey” was replaced by “by the French phytoplankton observation and monitoring network (Rephy)”

(5) Line 59 - 'resulting in' instead of 'resulting of'.

We agree with the reviewer and have replaced “resulting of” with “resulting in”.

(6) Line 65 - It seems that ', influencing the time of appearance of blooms' would be more correct than the current phrasing. The current phrasing is unclear regarding the relation between species, tolerance range, and the time of appearance of blooms.

To address this point, “Depending on the phytoplankton species, the tolerance range of physicochemical parameters is different and influences the time of appearance of blooms” was replaced by “Depending on the species of phytoplankton, tolerance to physicochemical parameters varies, which influences when blooms occur.”

(7) Line 76 - Run-on sentence which should probably be split after the reference to Wang et al., 2020.

We agree with the reviewer and have split the sentence.

(8) Line 89 - What are these observations?

This sentence was reformulated.

“Based on observations from the natural environment showing a potent relationship between Vibrio and Alexandrium algae bloom events, this study aim to determine in vitro, the main factors implicated in this relationship” was replaced by ”This study aims to describe observations made in the natural environment between Vibrio bacteria and Alexandrium algal blooms, and to determine in vitro the main factors involved in this relationship.”

(9) Line 94 - This is the first clear reference to a predator-prey interaction, and it is stated as if it's established. Is it not a central goal of the study to demonstrate that predation is even happening?

Based on the title and abstract, I would have expected the major claims of the paper highlighted in the abstract to be:

(i) that predation of algae by bacteria occurs in this system,

(ii) there is a social component of predation,

(iii) claims about what induces this predatory behaviour.

The summary has been amended accordingly, and the term “predation” has been removed, along with all sentences referring to it.

(10) Line 99 - What does n.d. mean?

This point was addressed in the revised version.

(11) Line 97 section - specify qPCR.

This point was clarified in the revised version.

(12) Line 139 - Mentioning the oligonucleotides in this part of the methods seems out of place. Would this not fit better in the section on Gene expression analysis?

This sentence was discarded from this paragraph.

(13) Line 147 - Where did the co-cultured phytoplankton species come from?

To answer this point, reference to Table 2 was added

(14) Line 149 - Is it known if the phytoplankton strains had all grown to the same density after 24 hours?

The doubling time of dinoflagellates in laboratory culture is between 5 and 7 days. During the duration of the experiments, the dinoflagellate concentration did not change significantly.

The sentence “(doubling time between 5 and 7 days)” was added

(15) Line 150 - Was the density of the Vibrio cultures at the different incubation times measured? Density might play an important role in predation, and so it would be important to control for density in these assays.

The concentrations of live vibrio in each individual culture were not actually measured. However, the role of vibrio density in attacks was measured and is shown in Figure 4A and observed in Fig 2B.

(16) Line 153 - How long was the co-incubation?

The incubation times were added in the revised version.

(17) Line 158 - What is mean by "independent experiments", more exactly?

To clarify this point, “Data are the means of three independent experiments” was replaced by “The data come from three independent experiments using independent phytoplankton cultures and independent bacterial cultures.”

(18) Line 161 - Perhaps give the source information about the Vibrio strain at its first mention.

A reference has been added in the revised preprint.

(19) Line 163 - line 141 refer to multiple non-axenic species, whereas here "the algal strain" is referred to.

And

(20) Line 164 - language phrasing throughout the manuscript could use some polishing, e.g., "this means that additional bacteria...".

To address this comment, “As the algal strain used in the study is not axenic, means that additional bacteria, other than the V. atlanticus LGP32, are potentially present in the experiments.” was replaced by “As the A. pacificum ACT03 strain (table 2) used in the study is not axenic, there is potential for bacteria other than V. atlanticus LGP32 to be present in the experiments.”

(21) Line 208 - Why were both magnitude and p-value criteria used rather than just p-values?

In the present proteomic approach each experimental condition was measured six times, and the average (mean) value was used to reduce random noise. Then we selected differences that had to be large enough to matter biologically, this is a central criterion and at least a 2-fold change was considered to focus exclusively on biologically relevant differences, which allowed us to control for the effect size. However, the differences also had to be statistically significant, we applied a statistical confidence at P < 0.01, to be sure that there is less than a 1% chance the result happened randomly. In the present proteomic approach each experimental condition was measured six times, and the average (mean) value was used to reduce random noise.

Then we selected differences that had to be large enough to matter biologically, this is a central criteria and at least a 2-fold change was considered to focus exclusively on biologically relevant differences, which allowed us to control for the effect size. However, the differences also had to be statistically significant, we applied a statistical confidence at P < 0.01, to be sure that there is less than a 1% chance the result happened randomly. We considered that using both criteria makes the results meaningful and trustworthy, not just a small or random fluctuation.

(22) Line 270 - Were these three replicate experiments also "independent"; if yes, in what sense?

“All experiments were conducted in triplicate” was replaced by “The experiments were performed using biological triplicates, each of which was analyzed in triplicate.”

(23) Line 296 - Perhaps "the temperature-sensitivity (or resistance) of" rather than "the nature of".

The modification was made in the new manuscript.

(24) Line 307 - The sentence mentions only one influential period that was removed from the dataset, but the word 'whenever' suggests multiple occurrences.

We agree, “whenever” was replaced by “because”.

(25) Line 325 - line 327 - The rationale behind the first part of the following sentence isn't clear to me, and what is meant by the second part is also not clear.

To clarify this point, “This result is consistent with the difficulty that Vibrio has in growing at temperatures below 20°C and with the complex interacting factors driving bloom dynamics (Laanaia et al., 2013)” was replaced by “This result is consistent with the difficulty Vibrio has in growing at temperatures below 20°C and with the many environmental factors that influence the dynamics of algae proliferation (Laanaia et al., 2013)."

(26) Line 327 - line 328 - Hard to interpret; does this refer to living algal cells, or all algal cells, living and degraded?

To improve clarity, “Interestingly, in spring 2015, the mean densities of all Alexandrium cells and of free-living Vibrio were positively correlated” was replaced by “Interestingly, in spring 2015, the mean densities of Alexandrium cells (living and degraded) and of free-living Vibrio were positively correlated”

(27) Figure 2 - These results strongly point to predation, but why the Vibrio population would already be elevated in the co-culture treatment relative to the control immediately after inoculation (0 hrs) is not clear.

The experiments were not conducted at the same time, and the first value on the graphs corresponds to the concentration of vibrio determined after 1 hour of exposure/incubation and not at time 0. Figures 2A and 2B have been modified accordingly, and substantial changes have been made to the relevant section of the results.

(28) Line 348 - There's no mention of Figure 2C in the main text, or of the statistical test associated with it in the Figure 2 legend.

To address this comment, Figure 2C has now been cited in the main text, and the statistical analysis method has been added to the Figure 2 caption.

(29) Line 352 - Text descriptions of videos are not easy to connect with the video content. Label the file names the same as how they are referred to in the text.

We agree with you, the sentence “Epifluorescence microscopy observation of GFP-labelled V. atlanticus LGP32 (previously grown in Zobell medium) in interaction showed that A. pacificum ACT03 cells that had lost their motility were attacked individually by V. atlanticus LGP32 before being lysed (Fig, 2C and Video 1). “was rephrased and replaced by “Epifluorescence microscopy observation of GFP-labelled V. atlanticus LGP32 (previously grow in Zobell medium) in interaction showed that V. atlanticus LGP32 simultaneously attacks A. pacificum ACT03 cells (Fig, 2C and Video 1).”

(30) Movie 1 could be cut to remove uninteresting footage at the start. What indicates lysis? Is the deformation of the cells an indication of lysis?

To respond to this comment, Video 1 has been shortened and in the caption, “degraded” was replaced by “lysed”

(31) Line 353 - Video could be zoomed in more on a few typical attacks to remove visual noise.

A chronological overview of an attack has been added to Figure 2 corresponding to Figure 2D, and a chronological overview of the overall event has been added to Figure 3 corresponding to Figure 3B1.

(32) Line 355 - There does not seem to be a Figure 3A2.

To address this point, the Fig. 2 and Fig. 3 has been revised for more clarity. See above

(33) Figure 3 - Can the authors fully exclude an effect of bacterial density as distinct from an effect of growth/starvation phase? It would be helpful to determine bacterial viable population densities at 12, 36, 60, and 126 hrs of incubation in Zobell medium, and to control for density in testing for effects on algae.

Information on Vibrio densities incubated in Zobell medium for 12, 36, 60, and 126 hours has been now included in the results section “Attack of A. pacificum ACT03 is activated by V. atlanticus LGP32 starvation.”

(34) Line 363 - It is unclear how the degradation of the flagella is apparent from movie 3. It would be helpful to have a comparison with healthy flagella.

Alexandrium cells with intact flagella move so quickly that it is impossible for us to follow them and film their flagella with the tools at our disposal.

For greater clarity, arrows have been added to videos 3, 4 and 5.

(35) Line 364 - Sudden change from referring to the recording as 'video' instead of movie. What is meant by erratic swimming? The cell does not seem to move much.

To address this comment, “Movie” was replaced by “Video” throughout the manuscript and “erratic swimming” was replaced by “irregular swimming”

(36) Line 365 - How did you observe the detachment of the flagellum?

The detachment of the flagellum can be observed using a confocal microscope. This process was filmed and presented in Video 3. Arrows have been added to the video to clearly indicate the flagellum detachment.

(37) Line 368 - Perhaps this is due to it not being clear regarding which movie is meant, but there is no clear attack visible in movie 4.

To make this clearer, arrows have been added to the video 4 to indicate attached cells.

And the sentence in the caption of the video 4 “Vibrio, filmed under a confocal microscope, attacks in groups one immobilized Alexandrium cell then moves on to attack — still as a group — another cell without touching the other whole cells, suggesting active communication between Vibrio cells” was rewritten and replaced by “This video, recorded under a confocal microscope, shows Vibrios simultaneously attacking a first immobilized Alexandrium cell, then moving on to attack a second cell without ever targeting the other cells present, suggesting active communication between the Vibrio bacteria.”

(38) Line 369 - It seems the peak attach % was reached at 45 minutes, not 15-30 minutes.

Sorry for the confusion. In fig. 3 for more clarity, the sentence “(A) Percentage of A. pacificum ACT03 motile cells. (B) cells attacked by V. atlanticus LGP32 and (C) cells lysis after 0, 15, 30, 45 and 60 min of interaction” was replaced by “(A) Cumulative percentage of motile A. pacificum ACT03 cells. (B) Cumulative number of cells attacked by V. atlanticus LGP32 and (C) Cumulative cell lysis after 0, 15, 30, 45 and 60 minutes of interaction.”

(39) Line 382 - "clearly show role of nutrient limitation", see comment re controlling for any role of bacterial density.

To address this point, information’s on Vibrio densities were added in the manuscript. See cf comment 33.

(40) Line 385 - line 386 - Phrasing unclear.

We have revised the text accordingly, “To this aim, A. pacificum ACT03 in exponential growth phase was first exposed for 30 min to supernatant from 60 hours starved V. atlanticus LGP32 Zobell media that induced 25% lysis of A. pacificum ACT03 cells and next to the corresponding V. atlanticus LGP32 cells. Group attacks were observed on non-degraded A. pacificum ACT03 cells, but not on lysed cells.“ was replaced by “To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 126-hour culture of V. atlanticus LGP32, which induced lysis of 70% of the A. pacificum ACT03 cells (Figures 3C and 3C1, arrow 2 and video 4). Next, cells of V. atlanticus LGP32 from a 60-hour culture, capable of attacking A. pacificum ACT03 cells (Fig. 3B), were added. For 1 hour of exposure, no attack was observed on the previously lysed algae.”

(41) Line 413 - Is this the only pathway for quorum sensing in V. atlanticus?

Indeed, the last two sentences of this paragraph are unclear.

To address this point:

“By targeted mutagenesis of key genes involved in QS pathways ΔluxM (HAI-1 production), ΔluxS (AI-2 production) and ΔluxR (high-density QS master regulator) did not lead to any change in the attack behaviour of V. atlanticus LGP32 (Fig. 4C).” was replaced by “Targeted mutagenesis of key genes involved in two of the three known QS pathways in vibrios (Fig. S3), ΔluxM (HAI-1 production), ΔluxS (AI-2 production), and ΔluxR (main high-density QS regulator), did not result in any changes in the attack behavior of V. atlanticus LGP32 (Fig. 4C).”

And “Taken together these results showed that attack by V. atlanticus LGP32 is not link to QS.” was replaced by. “Combined with the absence of overexpression of the CqsS gene (inducible by CAI-1) involved in the last known QS pathway in Vibrio (Fig. S3), these results indicated that the attack by V. atlanticus LGP32 is most likely unrelated to QS.”

(42) The references to tropism aren't clear.

You're right, there's no reason to use the term tropism here. We have removed it.

(43) Line 439 - Why was H3BO4 used as a control for the addition of FeCl3?

For clarity, the sentence “Boron being known to be a regulator or capable of being transported by vibrioferrin (Romano et al., 2013; Weerasinghe et al., 2013), we tested its potential involvement in the interaction but no effect was evidenced here.” was replaced by “Given that boron is known for its role in regulating a global bacterial cellular response to phytoplankton and to bind to vibrioferrin (Romano et al., 2013; Weerasinghe et al., 2013), we tested its potential involvement in simultaneous vibrio attacks. Compared to the Zobell control, no effect on the number of attacks was observed”

(44) Line 441 - line 449 - Should explicitly say in text that no attacks were observed for any species other than the Alexandrium and Gymnodinium species.

We agree and have explicitly stated in the text that no attacks were observed for any species other than Alexandrium and Gymnodinium.

(45) Line 454 - line 455 - The last part of this sentence seems a strange statement, since

(i) it has long been know that predatory bacteria can eat a wide range of eukaryotes, ii) one of the cited papers (Perez et al) actually highlights a case of bacterial predation on algae, and iii) in the next paragraph the authors themselves highlight Streptomyces predation of algae.

To make this clearer, « Among predators, predatory bacteria are found in a wide variety of environments, and like bacteriophages and predatory protists, they have been reported to prey exclusively on other bacteria » was replaced by “Among predators, predatory bacteria are found in a wide variety of environments and, like bacteriophages and predatory protists, feed primarily on other bacteria, although a few cases of predation on microbial eukaryotes have also been reported.”

(46) Line 455 - Better to clarify the authors' definition of a predator at the start of the paper. The offered definition seems more like a definition of 'consumer' than 'predator', as the latter normally involves both the killing and consumption of other organisms, not just consumption with some kind of "expense".

To address this comment:

- “predator behaviour” was replaced by “predator-like behaviour”

- and “Considering predator as a free organism that feeds at the expense of another, this study is the first evidence of the capacity of some Vibrio to develop a predatory strategy against an alga. This behaviour differs from parasitism, because the survival of Vibrio is not exclusively dependent on algae in environment” was replaced by “Consider a predator as a free-living organism that kills its prey and feeds on it, this study provides data suggesting the ability of Vibrios to develop an original predator-like behaviour to kill and feed on algae.”

(47) Line 457 - Don't see the benefit of trying to distinguish from parasitism here, especially since parasitism can be facultative, whereas the authors' phrasing suggests that it is always obligate.

You are right, this sentence has been deleted.

(48) Line 463 - line 464 - The authors should clearly explain exactly what detailed aspects of Myxococcus and Lysobacter predation they think the "attack stage" of V. atlanticus resembles.

Accordingly, “The second stage, the ‘attack stage’ corresponding to physical contact between Vibrio and Alexandrium resembles the ‘wolf-pack attack’ strategy described for Myxococcus xanthus and Lysobacter regardless of the prey species used, M. xanthus must be in close proximity to prey cells in order to induce their lysis and to benefit from their biomass (Martin, 2002; Perez et al., 2014)” was replaced by “The second stage, the ‘attack stage’ corresponding to the physical contact between Vibrios and Alexandrium, is similar to the strategy used by Myxococcus xanthus and Lysobacter. These bacteria must be in close proximity to their prey in order to cause lysis and utilize their biomass, regardless of the prey's species (Martin, 2002; Genovesi et al., 2013; Perez et al., 2016; Zhang et al., 2020)”

(49) Line 466 - line 467 - The comparison to bacteria clustering around lysed cells is surprising since the authors show that V. atlanticus does not attack already lysed cells.

The sentence was rephrased, “This phenomenon is comparable to that of bacteria clustering around lysed ciliate cells “was replaced by “Visually, this phenomenon resembles bacteria clustering around lysed ciliate cells.”

(50) Line 469 - Missing is a statement of exactly what criteria constitute "wolf-pack hunting behaviour" and exactly how V. atlanticus meets those criteria.

To address this point, “wolf-pack hunting behaviour” was replaced by “predator-like behaviour”

'Able of' should be corrected to 'Capable of'.

We agree and have reworded the sentence.

(51) Line 470 - Consider starting a new paragraph for the material on quorum sensing.

Accordingly, we have separated the section concerning QS pathway from the section concerning iron pathway.

(52) As part of their discussion on the role of iron uptake, can the authors comment on any relationship between starvation and iron uptake, and in particular the observations that, while general nutrient deprivation induces attacks, supplementation with a specific nutrient (iron) also induces attacks (Figure 4D)? Do bacteria starved for general growth substrates take up more iron than growing bacteria?

To respond to this comment, “Future study could demonstrate further the role of vibrioferrin in group attack, by adding iron-saturated vibrioferrin to algae-Vibrio co-cultures.” was replaced by “Interestingly, if a general nutrient deficiency causes attacks, iron supplementation increases the number of attacks (Figure 4D), suggesting the importance of iron absorption in the attack behavior. Future studies should determine whether nutrient deficiency increases the iron absorption capacity of Vibrios and whether this plays a major role in the attack mechanism.”

(53) Line 486 - Of what is boron known to be a regulator?

To respond to this comment, “Given that boron is known for its regulatory properties and for being transportable by vibrioferrin“ was replaced by “Given that boron is known for its role in regulating a global bacterial cellular response to phytoplankton and to bind to vibrioferrin”.