Hoverflies use a time-compensated sun compass to orientate during autumn migration

The sun is the most reliable celestial cue for orientation available to daytime migrants. It is widely assumed that diurnal migratory insects use a ‘time-compensated sun compass’ to adjust for the changing position of the sun throughout the day, as demonstrated in some butterfly species. The mechanisms used by other groups of diurnal insect migrants remain to be elucidated. Migratory species of hoverflies (Diptera: Syrphidae) are one of the most abundant and beneficial groups of diurnal migrants, providing multiple ecosystem services and undergoing directed seasonal movements throughout much of the temperate zone. To identify the hoverfly navigational strategy, a flight simulator was used to measure orientation responses of the hoverflies Scaeva pyrastri and Scaeva selenitica to celestial cues during their autumn migration. Hoverflies oriented southwards when they could see the sun and shifted this orientation westward following a 6 h advance of their circadian clocks. Our results demonstrate the use of a time-compensated sun compass as the primary navigational mechanism, consistent with field observations that hoverfly migration occurs predominately under clear and sunny conditions.

Minor points: -Line 71: "is has been" should be "it has been" -Lines 143-147: to the best of my knowledge, the LEE quarter white diffuser effectively cuts out all the polarized light in the UV range. Are the hoverfly's polarization detectors UV sensitive (as in other dipterans)? -Line 184: Sun's azimuth should be sun's azimuth -Line 227: "being the azimuth" should be "being the sun's azimuth"? -Line 450: A reference regarding polarization orientation in locusts is missing.

20-May-2021
Dear Dr Wotton: I am writing to inform you that your manuscript RSPB-2021-0936 entitled "Hoverflies use a timecompensated sun compass to orientate during autumn migration" has, in its current form, been rejected for publication in Proceedings B.
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Sincerely, Dr Locke Rowe mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Two reviewers have seen your interesting manuscript and as you will see both are very positive. However, both also have a number of substantial questions and comments, and both suggest possible new experiments to tighten the conclusions of the paper. For instance, reviewer 1 would like to have seen a control to establish that the sun is indeed used as a compass (e.g. by simulating overcast conditions or by shifting the position of an artificial sun). Both were also a bit worried about the possibility of cue conflicts. One thing that occurred to me that might go a long way to satisfying Reviewer 1 is that you have flown (non-time-shifted) hoverflies from morning to afternoon under the natural sun and have already quite a number of flight tracks. You could compare the mean direction of a population of hoverflies flown between (say) 10:30 and 11:30 with the mean direction of a population of hoverflies flown between (say) 15:30 and 16:30 (when the sun has traversed across the sky). If the mean directions are statistically indistinguishable then this would be very strong evidence for the use of the sun (or its polarisation pattern, or both) as a time-compensated compass. From your data I suspect this will be the case. At any rate, prior to publication, the comments and criticisms of the two reviewers should be adequately addressed.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) Please find my review in the attached pdf file.
Referee: 2 Comments to the Author(s) In the paper "hoverflies use a time-compensated sun compass to orientate during migration" by Massy et al., the authors performed behavioral experiments to study if hoverflies employ a timecompensated sun compass during their migration. To study this, the authors tethered individual hoverflies at the center of a flight simulator setup and allowed them to set the desired direction with respect to the sky. Without any manipulation, the hoverflies maintained highly directed flights in the expected southerly direction. However, when the authors clock-shifted a population of hoverflies by 6 hours (they delayed the perceived beginning of the day) and tested them in their flight simulator, the hoverflies kept heading directions with respect to the sky that roughly matched the perceived time of day (morning) rather than the actual time of day (afternoon). This suggests that the hoverflies use a time-compensated sky compass for orientation. In addition, the authors generated a model that allowed them to calculate how efficiently the predicted southward direction was kept by the clock-shifted hoverflies. The behavioral data shown in Fig. 2 are solid and suggest that hoverflies rely on a timecompensated sky compass for orientation. I found the paper an easy read but have difficulties with some points. Please, see below my comments. I hope they will help to improve the paper/ to clarify some misunderstandings. Major points: -In general, I have difficulties with the term "partially" time-compensated compass. Why should an animal only "partially" compensate for the position of the sun? In my opinion, this does not make full sense from a biological point of view, but I am happy to discuss this with the authors in a potential revised version of the manuscript.
-In my opinion, the authors over-interpret the 70° difference between the direction taken by animals without any manipulation and the clock-shifted animals. The aspect that the hoverflies changed their heading by about 70° (rather than e.g. the expected 90° for a linear model) could, for instance, have methodical reasons, such as the tethering or the way they were kept under artificial light. I think an elegant control would have been to keep hoverflies under the same conditions (artificial light) without any clock shift. In addition, the 70° could be based on a cue conflict situation that the authors created through the time shift (e.g. sun position vs. the earth magnetic field). Given that the data show a high variance, I would be more careful with the interpretation of the change in heading. For instance, what would happen if the authors recalculated the headings in fig. 2b according to the perceived time of the clock-shifted animals? Does it look like the plot in 2a? Are the data significantly different from the data in 2a? Again, I am happy to discuss this in a potential revised version of the manuscript but in my opinion, the mean direction, combined with a high CI, is not very meaningful. I believe an interpretation of the changes in direction would require additional experiments (e.g. shifting the animals by -3h and/or advancing their circadian clock by 3/6 hours and testing the changes in heading).
-I believe the model requires some more explanation in the results. I find it difficult to understand what exactly the authors would like to show with the model. What does the efficiency of 0.6 mean? To me, it seems as if this would be efficient to roughly migrate southwards if an animal does not have a specific migration destination. What would the difference between 0.6, 0.98, and 1 efficiency mean in distance per day? In addition, why did the authors not use the actual 188.2° as their expected direction for the model? -This might be semantic but why do the authors call it a sun compass? The hoverflies could for instance use the polarization pattern as their main orientation reference. Given that the dominant skylight cue is not known in hoverflies, I would refer to it as a "time-compensated sky compass". Minor points: -Line 71: "is has been" should be "it has been" -Lines 143-147: to the best of my knowledge, the LEE quarter white diffuser effectively cuts out all the polarized light in the UV range. Are the hoverfly's polarization detectors UV sensitive (as in other dipterans)? -Line 184: Sun's azimuth should be sun's azimuth -Line 227: "being the azimuth" should be "being the sun's azimuth"? -Line 450: A reference regarding polarization orientation in locusts is missing.

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author Well done, nice manuscripts.

27-Aug-2021
Dear Dr Wotton I am pleased to inform you that your Review manuscript RSPB-2021-1805 entitled "Hoverflies use a time-compensated sun compass to orientate during autumn migration" has been accepted for publication in Proceedings B.
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31-Aug-2021
Dear Dr Wotton I am pleased to inform you that your manuscript entitled "Hoverflies use a time-compensated sun compass to orientate during autumn migration" has been accepted for publication in Proceedings B.
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Thank you for your fine contribution. We would like to thank both you and the reviewers for the helpful comments. We believe we have been able to address all of these and below we set out our responses in blue together with a summary of the comments addressed.
In brief, we present additional evidence that (1) migratory hoverflies undertake directed flight and that this group orientation increases in flights undertaken later in the day; (2) that hoverflies use the sun as their primary cue; and (3) that our hoverflies are indeed clock-shifted.
However, this work has substantially increased the length of our paper beyond the 10-page limit of Proceedings B and we have therefore chosen to remove the model describing the efficiency of southwards migration under varying degrees of time-compensation. It is our intention to resubmit this work as a standalone paper to Biology Letters in due course.
Despite this, our much improved paper remains the first confirmation of the use of a timecompensated sun compass for migration in insects outside of a select few lepidopteran species, and therefore provides strong support for its repeated co-option, hypothesised to have occurred during the evolution of migration in different lineages.
I hope you agree that this update has substantially enhanced the manuscript and we look forward to your response.

Associate Editor Board Member
Editor comment: One thing that occurred to me that might go a long way to satisfying Reviewer 1 is that you have flown (non-time-shifted) hoverflies from morning to afternoon under the natural sun and have already quite a number of flight tracks. You could compare the mean direction of a population of hoverflies flown between (say) 10:30 and 11:30 with the mean direction of a population of hoverflies flown between (say) 15:30 and 16:30 (when the sun has traversed across the sky). If the mean directions are statistically indistinguishable then this would be very strong evidence for the use of the sun (or its polarisation pattern, or both) as a time-compensated compass. From your data I suspect this will be the case.
Our reply: We thank the editor for this excellent suggestion that led us to explore deeper into the time-associated variation in our data. Unfortunately, the suggested analysis was not possible as we have only 3 datapoints for each of these time windows ( Figure 2d).

Our reply continued:
We also provide additional data, including back-transformed clock-shift data, as suggested by referee 2, and analysis of the last 15 flown hoverflies that provides stronger support for the clock-shift results (see text below).

New manuscript text:
As the clock-shifted experiments were undertaken later in the day when the sun would naturally be in a more clockwise position, to rule out simple phototaxis, the analyses were repeated on angles that were rectified to be relative to the direction of the sun, so that the direction of the sun is in the 180° position ( Figure S1)   been seen in several groups (crustacean sandhoppers: Wallraff, 1981;honeybees: Gould, 1980;pigeons: Schmidt-Koenig et al. 1991a;Wiltschko et al. 1994;Chappell, 1997), for example desert ants underestimate the rate of movement of the sun's azimuth when it is high and overestimate this rate of movement when it is low (Wehner, 1984). We have attempted to clarify this in the manuscript (see comments below).

Referee comment:
In my opinion, the authors over-interpret the 70° difference between the direction taken by animals without any manipulation and the clock-shifted animals. The aspect that the hoverflies changed their heading by about 70° (rather than e.g. the expected 90° for a linear model) could, for instance, have methodical reasons, such as the tethering or the way they were kept under artificial light. I think an elegant control would have been to keep hoverflies under the same conditions (artificial light) without any clock shift.
Our reply: We agree with the reviewer and have clarified this at various points in the text. We now state in the results: "The mean direction of the time-shift experiment was 68.9° clockwise from that of the sun-compass experiment, representing 70% of the 98.1° adjustment required to fully compensate for the mean six-hour azimuth change, although the confidence interval does not rule out complete time compensation." In addition, we have added new clarifying text to the discussion: in migrating hoverflies, a finding also supported by the lack difference between mean directions in hourly tracks and headings of radar detected hoverflies throughout the entire day.

Referee comment:
In addition, the 70° could be based on a cue conflict situation that the authors created through the time shift (e.g. sun position vs. the earth magnetic field). Given that the data show a high variance, I would be more careful with the interpretation of the change in heading. For instance, what would happen if the authors recalculated the headings in fig. 2b according to the perceived time of the clock-shifted animals? Does it look like the plot in 2a? Are the data significantly different from the data in 2a? Again, I am happy to discuss this in a potential revised version of the manuscript but in my opinion, the mean direction, combined with a high CI, is not very meaningful. I believe an interpretation of the changes in direction would require additional experiments (e.g. shifting the animals by -3h and/or advancing their circadian clock by 3/6 hours and testing the changes in heading).
Our reply: We thank the referee for bringing up this interesting possibility. We have investigated this further in relation to the magnetic tethering system used in our experiments. In our system the hoverfly is exposed to a symmetrical magnetic field of more than 400 gauss (https://www.kjmagnetics.com/magfield.asp?pName=RX8CC). We believe this makes it highly unlikely that the weak magnetic field emanating from the Earth's surface of 0.25 to 0.65 gauss would be able to provide a suitable conflicting cue. In addition, we have carried out the suggested back transformation of the clock-shifted data and show that the sun compass and back transformed data are not significantly different, supporting our initial interpretation that the hoverflies are clockshifted. The modified results section is copied below.
New manuscript text: As the clock-shifted experiments were undertaken later in the day when the sun would naturally be in a more clockwise position, to rule out simple phototaxis, the analyses were repeated on angles that were rectified to be relative to the direction of the sun, so that the direction of the sun is in the 180° position. (Figure S1)  (Figure 3a-c) and compensating for its position rather than just following its course throughout the day.
Our reply continued: As suggested we have tried to provide a more careful interpretation of our findings, discuss potential sources of error and suggest follow up experiments that may help to clarify the findings (see new text presented in response to previous comment). Regarding the high confidence intervals, we agree that they are not ideal, however we disagree that combined with the mean direction that they are not very meaningful. The mean directions are statistically significant as confirmed by a Mardia-Watson-Wheeler test, and this significance is increased when analysing the last half of the data set (p-value changes from 0.02 to 0.0089) and the rectified last half of the data set (p-value from 0.02 to 0.0043). In addition, the confidence intervals are reduced in the analysis of the last half of the data set from 71° to 53°, approaching the 36° seen by Mouritsen & Frost for the monarch butterfly. We believe that this, together with the new analysis of the back transformed clock-shift data, indicates that the hoverflies are indeed clock-shifted, were flying in different directions with respect to the sun and compensating for its position rather than just following its course throughout the day. Finally, while additional clock-shift experiments would be a nice addition and may help, with the right set up, to distinguish between full, partial, and time-averaging methods, we do not believe that it is necessary to demonstrate that the sun compass is timecompensated as was our goal here. Referee comment: This might be semantic but why do the authors call it a sun compass? The hoverflies could for instance use the polarization pattern as their main orientation reference. Given that the dominant skylight cue is not known in hoverflies, I would refer to it as a "time-compensated sky compass".
Our reply: We agree with the referee on this point given the evidence we provided in the original version. However, our new manuscript includes a 'restricted view' experiment that demonstrates loss of group orientation when the sun is obscured but other cues such as intensity gradients, chromatic gradients and polarization of light remain. We believe this clearly demonstrates that the sun is the dominant skylight cue and therefore the term 'sun compass' is now accurate.
Referee comment: Line 71: "is has been" should be "it has been"

Our reply: Done
Referee comment: Lines 143-147: to the best of my knowledge, the LEE quarter white diffuser effectively cuts out all the polarized light in the UV range. Are the hoverfly's polarization detectors UV sensitive (as in other dipterans)?
Our reply: Unfortunately, we did not measure light in the UV range, and we are unaware of any measures of the sensitivity of hoverfly polarization detectors in the dorsal rim area of the eye. We note however, that while this signal may have been attenuated in the sun compass experiment, it was not in the restrictor experiments that were carried out with an open lid. Therefore, while we cannot rule out a role for polarized light in combination with the sun, it is not sufficient by itself for orientation in a seasonally beneficial direction. We now cover these points in the modified text: New manuscript text: In the field we observed a pattern of surges of migratory activity during sunny periods, with little activity when the sun was obscured. This observation, together with the loss of group orientation seen in our restricted-view experiment, suggest that the sun acts as the primary celestial cue for orientation and that hoverflies wait for favourable navigational conditions, namely a visible sun, to migrate rather than relying on other cues. However, individuals flown with a restricted view are still able to maintain directed flights, as indicated by high individual r-values, but these are arbitrary with respect to the celestial cues and may represent an ancestral mechanism in insects for covering large distances [51,52]. To further investigate this, future studies should determine the contribution, if any, of other celestial cues attenuated in our sun compass experiments such as intensity gradients, chromatic gradients or the polarization of light for orientation, as these may work in concert with the sun, despite being insufficient by themselves for group orientation in our experiments.
Referee comment: Line 184: Sun's azimuth should be sun's azimuth

Our reply: Done
Referee comment: Line 227: "being the azimuth" should be "being the sun's azimuth"?
Our reply: Done