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  1. Evaluation Summary:

    This manuscript reports a new method for high-throughput analysis of C. elegans feeding behaviour that overcomes shortcomings of existing methods. It is a useful technique that will be interesting for scientists studying feeding dynamics in worms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this paper Bonnard et al. describe the development of a new method to quantify C. elegans feeding behaviour. The circuit mechanisms underlying feeding behaviour of worms are not fully understood and studying them potentially can lead to fundamental new insights into the control of rhythmic behaviours in general. In classical work, the up to 5Hz peristaltic movements of the worms' pharynx were assessed manually, a painful procedure which is challenging and error prone. Alternative methods have been developed, either by video recording and subsequent slo-mo manual analysis, or recording of electropharyngeogram in microfluidic devices, however, these prevent observations in unrestrained behaving animals. The approach reported here is based on a sophisticated image processing pipeline analysing freely moving worm populations of animals with fluorescently labelled pharynges. The authors convincingly show that peristaltic pumping rates can reliably be measured across development, under various feeding conditions and capturing known and previously undescribed phenotypes of feeding mutants. The method represents a significant improvement and possibly seems applicable to researchers in the community; however, it cannot measure detailed aspects of pharyngeal dynamics like it is possible with electropharyngeogram recordings. I see great potential also to perform long-term recordings, this unfortunately is not addressed in the current manuscript.

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  3. Reviewer #2 (Public Review):

    Feeding behaviour in C. elegans has been extensively studied over decades. Several methods of measuring feeding exist, but none can directly measure both pumping and locomotion behaviour in freely-moving worm populations. The authors have developed a new imaging-based method for automated detection of pharyngeal pumping events in freely moving C. elegans populations, and can thus simultaneously measure pumping and locomotion behaviour in tens of worms, at a single-worm, single-pump resolution that is not possible with previous methods. This user-friendly method can be applied to several research directions, such as large-scale foraging, behavioural coordination, and high throughput screening.

    The authors designed their new method to be broadly applicable and user-friendly, for easy adaptation in other research labs. However, adding direct evidence to show that "the method is relatively insensitive to the optical instrument used" will better support this claim of wider application.

    The authors carefully benchmarked their new method against expert annotations and existing results from previous methods, to both validate their method and reveal additional advantages. They also assessed potential pitfalls of the method such as by examining the effect of fluorescence imaging on the behavioural outcome, albeit only at the timescale of minutes. The effect of longer-term fluorescence imaging should be further explored, which is relevant for large-scale foraging experiments that the authors discussed. It could be helpful to determine the maximum total exposure for the method to still be valid, both in terms of pump detection (which could be sensitive to photobleaching) and behavioural modulation (which could be sensitive to higher phototoxicity).

    Overall, the manuscript is well-written and the results are clearly presented both in terms of statistics and interpretation. Methodological details are well-documented and openly accessible.

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  4. Reviewer #3 (Public Review):

    In this manuscript, the authors present a method for simultaneous assessment of pharyngeal pumping (feeding) and locomotion in many C. elegans simultaneously. In this technique, imaging of the fluorescent labeled pharynx provides a measure of velocity and pumping rate, through analysis of the spatial variations in fluorescence.

    The technique is clearly described, well-validated, and yields some novel results. It has the advantage that it can be performed using microscopes found in many C. elegans laboratories.

    Some limitations of the method include its reliance on fluorescence imaging, which is a hindrance to genetic analysis, computational intensiveness, and phototoxic effects of fluorescence excitation that are not fully explored in the manuscript.

    The authors show the utility of their method by assessing pharyngeal pumping and motor behavior (1) during development, (2) in the presence or absence of food, and (3) in the presence of two mutations affecting feeding.
    Although I understand these are proof-of-principle demonstrations, I still came away feeling underwhelmed by these examples. I did not see any results here that could not have been obtained fairly easily with conventional techniques.

    Given these limitations, I feel the method's eventual impact in the field will be relatively small.

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  5. Author Response

    Reviewer #2 (Public Review):

    Feeding behaviour in C. elegans has been extensively studied over decades. Several methods of measuring feeding exist, but none can directly measure both pumping and locomotion behaviour in freely-moving worm populations. The authors have developed a new imaging-based method for automated detection of pharyngeal pumping events in freely moving

    C. elegans populations, and can thus simultaneously measure pumping and locomotion behaviour in tens of worms, at a single-worm, single-pump resolution that is not possible with previous methods. This user-friendly method can be applied to several research directions, such as large-scale foraging, behavioural coordination, and high throughput screening.

    The authors designed their new method to be broadly applicable and user-friendly, for easy adaptation in other research labs. However, adding direct evidence to show that "the method is relatively insensitive to the optical instrument used" will better support this claim of wider application.

    We appreciate the reviewer’s suggestion to show evidence that our method will also work on data acquired on different microscopes. We now present data obtained on a second epi-fluorescent microscope, which was downscaled and analyzed in Fig. 1H-J.

    The authors carefully benchmarked their new method against expert annotations and existing results from previous methods, to both validate their method and reveal additional advantages. They also assessed potential pitfalls of the method such as by examining the effect of fluorescence imaging on the behavioural outcome, albeit only at the timescale of minutes. The effect of longer-term fluorescence imaging should be further explored, which is relevant for large-scale foraging experiments that the authors discussed. It could be helpful to determine the maximum total exposure for the method to still be valid, both in terms of pump detection (which could be sensitive to photobleaching) and behavioural modulation (which could be sensitive to higher phototoxicity).

    We thank the reviewer for this comment. In response to their comment and related comments by the other reviewers, we have provided bleaching curves and evidence of long-term imaging to show the potential of the methods for longer scale assays. We found that with our illumination intensity (see methods), bleaching was significant at a time scale of ~1h. We then added triggered illumination and could extend the recording time to ~5 h (Methods). Additionally, we perform a supplementary control for viability of worms exposed to continuous light (not triggered) for 5 hrs. We do not observe any apparent phototoxic effect.

    Overall, the manuscript is well-written and the results are clearly presented both in terms of statistics and interpretation. Methodological details are well-documented and openly accessible.

    We thank the reviewer for their positive view of our work and their appreciation for our efforts to document both data and software.

    Reviewer #3 (Public Review):

    In this manuscript, the authors present a method for simultaneous assessment of pharyngeal pumping (feeding) and locomotion in many C. elegans simultaneously. In this technique, imaging of the fluorescent labeled pharynx provides a measure of velocity and pumping rate, through analysis of the spatial variations in fluorescence.

    The technique is clearly described, well-validated, and yields some novel results. It has the advantage that it can be performed using microscopes found in many C. elegans laboratories.

    We appreciate that the reviewer recognizes the wide applicability of the method across many C. elegans laboratories.

    Some limitations of the method include its reliance on fluorescence imaging, which is a hindrance to genetic analysis, computational intensiveness, and phototoxic effects of fluorescence excitation that are not fully explored in the manuscript.

    The authors show the utility of their method by assessing pharyngeal pumping and motor behavior (1) during development, (2) in the presence or absence of food, and (3) in the presence of two mutations affecting feeding.

    Although I understand these are proof-of-principle demonstrations, I still came away feeling underwhelmed by these examples. I did not see any results here that could not have been obtained fairly easily with conventional techniques.

    We appreciate the constructive criticism of the reviewer and highlight in the revised version the fact that using conventional techniques such studies would require tens of hours of experiment time. We would like to emphasize the comparisons in Table 1 where we show other methods and their current limitations. Obtaining a dataset such as in Figure 3 which comprises a total of 34 worm-hours of pumping observation from unrestrained animals is to our knowledge currently impractical with competing methods. We would like to remind the reviewer that, using our method we were able to reveal bimodal distributions within a population as illustrated, for instance, in Fig. 3F, 4B, and 4F. These observations are not possible when the single worm resolution is not accessible or when large statistics are not feasible as it happens with previous methods.

    Given these limitations, I feel the method's eventual impact in the field will be relatively small.

    In this study, we present a method allowing performing behavioral studies on worm populations at high throughput and reduced costs. Such a technique opens the door to many laboratories that can not do EPG recordings or microfluidics due to the technical difficulties, or that want to study animals in their normal plate context. We also would like to emphasize that there are already more than 1500 strains containing myo-2 promoter transgene available on CGC, which would be amenable to our imaging approach. These transgenic strains form broad classes of interest, such as thermotolerance, ER stress resistance, aging and neural-circuit specific genes.

    Pharyngeal pumping has also been used as a read-out for pharmacological screens, for example, bacteria pre-loaded with pharmacological agents are tested for their effect on pharyngeal pumping rate. Pharaglow offers a high-throughput and sensitive method to measure the pumping rate. This will benefit the field who use C. elegans pumping for pharmacological screens, and pave the way for the researchers who plan to use but are hindered by existing techniques.

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