Do Glycogen Content and Thermal Preference in Worker Bees Vary Depending on Geographical Origin? A Comparison of Carniolan Honeybees, Central European Honeybees and Caucasian Honeybees

Round 1

Reviewer 1 Report

The authors presented a work on glycogen content and thermal preferences in worker honey bees of different geographical origin (i.e., Carniolan, Central European and Caucasian). The authors also compared the glycogen content and the thermoregulatory behaviour of summer bees and bees preparing for wintering. They found a difference in the glycogen content related to both season and bees origin. Particularly, glycogen was higher in winter bees than in the summer ones in the Carniolan and Central European subspecies, while the opposite occurred in the Caucasian Honey bee. Differences were not detected in the thermal preference.

Overall, the work is of scientific interest and can make a contribution to our knowledge of the different adaptation of honey bee subspecies to different climate conditions, especially in view of the climate changes already underway.  However, improvements would increase the quality of the work. My major concern is the sample size used for the thermal preference experiment. I am not familiar with this type of experiment, but 6-4 bees seem too small to produce significant results. Following are my comments: 

Introduction

- Reading the paragraph on vitellogenin and Juvenile hormone one wonders why the authors did not also investigate these parameters, which are equally important for the longevity and wintering of bees. Is there a particular reason? Do the authors plan to conduct future experiments on this? 

- line 51: "It occurs at high levels, and the Juvenile hormone levels are low in bees preparing for wintering, while the opposite is true in summer bees". This sentence is not clear, please edit making clear which occurs at high levels.

 Materials and methods

- Why was glycogen measured in whole bee bodies instead of in muscle and fat bodies? As the authors state in the discussion, this may have affected the results.

- For measurement of thermal preference 16 bees were used: 6 Central European, 6 Caucasian and 4 Carniolan. I assume that these groups were further divided in half resulting in 3 summer bees and 3 wintering bees per subspecies (2 and 2 for Carniolan). Is this the case? I suggest specifying more clearly the methods and the number of replicates/bees used. The sample size of this experiment is my major concern. I have never done this kind of experiment, but 2-3 bees per group seem too few to me. Why weren't more individuals used? Did the authors make a power analysis to ascertain the appropriate sample size? Furthermore, although the authors had already studied the thermoregulatory behaviour of the Carniolan bees several times, I think they should have used the same number of bees as the other two subspecies. 

-  Is there a reference for the glycogen measurement method? Please, provide it. Same suggestion for the thermoregulation experiment.

- Line 120: please, provide a description of the chambers used for the thermal preference recording. 

- Line 123: which food was supplied to bees?

- The method of measuring thermal preference is not clearly explained. How many days did the experiment last? The position of the bees in the chambers was recorded for how long? What is the number of observations? Please explain in more detail. 

- Concerning the statistical analysis, did the authors consider models? Since the effect of multiple factors (season and subspecies) on a parameter is investigated, I think a better analysis could be achieved with a model. With a model, it can be better observed whether the effect of subspecies or bee season on the measured parameters is stronger.

Results

- Data of average glycogen content have a very high standard deviation. The authors should consider and discuss this. 

- In figures 3 and 6, it should be specified in the caption what the white and black columns represent. 

Discussion

- Line 214-216: I think this is a repetition of the introduction, which is not very functional for the discussion.

- Line 216-224: this whole paragraph should be shortened. 

- In general, more bibliographical references could be included. Are there other studies to support the findings?

Author Response

Response to Reviewer 2 Comments

Thank you very much for your valuable comments. We tried to respond to all of them and modify and correct the text in accordance with the recommendations. We have also made some auto-corrections to improve the readability and understanding of our manuscript.

Point 1: L 18. rewrite - mention two seasons

Response 1: Corrected as suggested by adding the word "autumn" next to "bees preparing for wintering"

Point 2: L 19. .....: Honeybees---- honeybees --- applicable in many parts of the manuscript. -

Response 2: We corrected accordingly, consequently using Honeybee with a capital letter everywhere

Point 3: L 19-20. glycogen content in summer bees? mention clearly -

Response 3: For better clarity, throughout the whole manuscript, we changed

“bees preparing for wintering”

to

“autumn bees preparing for wintering”

leaving unchanged “summer bees”.

Point 4: L 23. Keywords--- arrange alphabetically. -

Response 4: Corrected accordingly

Point 5: L 26. According to their........ distinguished. ---- whose? honeybees. Who distinguished? add references. -

Response 5: We added the word “Honeybee” and relevant references: [1-4]

Point 6: L 76-77. Rewrite -

Response 6: Rewritten accordingly:

“Equally important is the storage of glycogen in individual bee bodies.”

Point 7: L 89. ... different geographical areas. --- mention the areas. -

Response 7: We provided geographical data on the origin:

“The subspecies used in Poland come from different geographical areas, and only the Central European Honeybee is native to Poland. The Carniolan bee originally inhabited a large part of Europe east of the Alps and the Western Carpathians and south of the forest-steppe zone to the Black, Aegean and Adriatic Seas. The Caucasian honey bee originated in the higher parts of the Greater and Lesser Caucasus Mountains. This subspecies has spread to Russia, Poland and North America. In turn, Central European Honeybee inhabited Western and Central Europe areas. The breed's purity occurs in France, Poland and the Alps [9].”

Point 8: L 101-103. days old.--- How the authors determined the age?

Response 8: We provided the necessary data to the manuscript:

“When collecting bee samples, we were guided by the colour of the bees, collecting the youngest "grey bees", which have characteristic hair that wears off over time. Therefore, the individuals participating in the experiment could not be more than 3-6 days old in the imago stage.”

Point 9: L101-103: thermoregulatory behaviour----- need more detail on how they studied.

Response 9: We provided the necessary data to the manuscript. We removed the incorrect sentence

"The Carniolan Honeybees were represented by four individuals only because we studied thermoregulatory behaviour many times before [11-15]."

We only reported the sample size for Carniolan Honeybee. We moved the references regarding our previous studies on the thermoregulatory behaviour of worker bees of the Carniolan bee to the relevant paragraph in the Material and Methods chapter. We added to this paragraph more detailed information on the construction and conduct of tests using temperature gradient chambers and a figure illustrating that:

“We conducted present studies according to the methodology developed and used in our previous research [11-15].". Fig. 1 presents the diagram of the experimental setup. To determine the temperature preferred by the bees, we placed them in an aluminium chamber (0.60 x 0.034 x 0.022 m), one of which was one end heated (by a built-in thermostat), and the other was cooled by a cryostat K21 E20 (GK Sondermaschinenbau GmbH – 103 Labortechnik Medingen). That generated a linear temperature gradient along the entire length of the chamber, ranging from 10-50°C. Due to temperature perception ability, the bees occupied the most advantageous (most appropriate) places in the thermal gradient chamber.”

Point 10: L 103. [11-15]

Response 10: Corrected accordingly

Point 11: L 117. anthrone reagent---- needs more detail, including concentration.

Response 11: We provided more data to the manuscript:

“Glycogen concentrations in the body of Honeybee worker bees affiliated with the studied subspecies were determined by absorption spectrophotometry using anthrone reagent [12-14]. The anthrone reagent was prepared on the day of the assay by dissolving 100 mg of anthrone in 50 ml of 95% sulfuric acid.”

Point 12: L 148. p<0.0014---- p<0.01  -

Response 12: Corrected accordingly

Point 13: L 149. g.b.w.--- use full form initially. -

Response 13: Corrected accordingly:

“= micrograms per one gram of body weight”

Point 14: L 160, 167. p<0.0000----- p<0.001  -

Response 14: Corrected accordingly

Point 15: Figures 3 & 6. black bars for spring? mention clearly. -

Response 15: Corrected accordingly:

“Figure 34. Mean glycogen content (in μg/g.b.w.) in the organisms of worker bees of the subspecies studied in autumn (white bars) and spring (black bars). Statistical significance was determined (*p < 0.05; **p < 0.01; ***p < 0.001).”

Point 16: L 194. Fig. 4--- Figure 4 -

Response 16: Corrected accordingly

Point 17: L 202 p<0.154----- p= 0.15  -

Response 17: Corrected accordingly

Point 18: References: maintain the journal's style. -

Response 18: We do our best to maintain the journal’s style

Point 19: I suggest adding another section, 'Conclusion,' after the Discussion section.

Response 19:Section ‘Conclusion’ was added to the manuscript

“5. Conclusions

In this study, we determined the glycogen content in the bodies of three varieties of honey bees: Carniolan, Caucasian and Central European. We also studied their thermal preferences. We conducted the experiments on two groups: autumn bees pre-paring for wintering and summer bees. We found that:

1) Bees' subspecies affiliation, and thus their geographic origin, affects their glycogen concentration;

2) The time of year in which honey bee workers are born determines the glycogen content of their bodies;

3) Neither the geographic origin nor the season in which they were born has an influence on the preferred temperature.

4) There is a discrepancy between the Caucasian bees and the remaining subspecies in terms of the glycogen content differences between the group of autumn bees preparing for wintering and the group of summer bees.”

Reviewer 2 Report

The work is valuable in the management of honeybees. However, representation is poor. The material and method section needs to improve. I suggest adding another section, 'Conclusion,' after the Discussion section. Authors can also see the attached PDF file for specific comments.

L18: rewrite - mention two seasons.

L19,.....: Honeybees---- honeybees --- applicable in many parts of the manuscript.

L19-20: glycogen content in summer bees? mention clearly.

L23: Keywords--- arrange alphabetically.

L26: According to their........ distinguished. ---- whose? honeybees. Who distinguished? add references.

L76-77: rewrite.

L89: ... different geographical areas. --- mention the areas.

L91: 3-6 days old.--- How the authors determined the age?

L101-103: thermoregulatory behaviour----- need more detail on how they studied.

L103: [11-15]

L117: anthrone reagent---- needs more detail, including concentration.

L148: p<0.0014---- p<0.01

L149: g.b.w.--- use full form initially.

L160, 167: p<0.0000----- p<0.001

in Figures 3 & 6--- black bars for spring? mention clearly.

L194: Fig. 4--- Figure 4

L202: p<0.154----- p= 0.15

References: maintain journal's style.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

Thank you very much for your valuable comments. We tried to respond to all of them and modify and correct the text in accordance with the recommendations. We have also made some auto-corrections to improve the readability and understanding of our manuscript.

Point 1: My major concern is the sample size used for the thermal preference experiment. I am not familiar with this type of experiment, but 6-4 bees seem too small to produce significant results.

Response 1: We agree with the Reviewer. However, to the best of our knowledge, the sample size we used represents enough minimum to produce significant results. We will discuss this further in Point 5.

Point 2: Reading the paragraph on vitellogenin and Juvenile hormone one wonders why the authors did not also investigate these parameters, which are equally important for the longevity and wintering of bees. Is there a particular reason? Do the authors plan to conduct future experiments on this?

Response 2: We thank the Reviewer for this commentary. Yes, We plan to conduct such experiments. In a comparative aspect, we want to investigate vitellogenin and Juvenile hormone and analyse the bees' lifetime in the thermal gradient.

Point 3:  "It occurs at high levels, and the Juvenile hormone levels are low in bees preparing for wintering, while the opposite is true in summer bees". This sentence is not clear, please edit making clear which occurs at high levels.

Response 3: Corrected accordingly:

“In the autumn bees preparing for wintering, Vitellogenin occurs at high levels, and the Juvenile hormone levels are low, while the opposite happens in summer bees [8].”

Point 4: Why was glycogen measured in whole bee bodies instead of in muscle and fat bodies? As the authors state in the discussion, this may have affected the results.

Response 4: We agree with the Reviewers. This investigation is preliminary. As we state in the Discussion:

“These were preliminary analyses aimed at determining general relationships. We plan to perform analogous comparative studies of the same material, which will concern individual isolated organs of the bee body: head, flight muscles, and fat body.”

Point 5: For measurement of thermal preference 16 bees were used: 6 Central European, 6 Caucasian and 4 Carniolan. I assume that these groups were further divided in half resulting in 3 summer bees and 3 wintering bees per subspecies (2 and 2 for Carniolan). Is this the case? I suggest specifying more clearly the methods and the number of replicates/bees used. The sample size of this experiment is my major concern. I have never done this kind of experiment, but 2-3 bees per group seem too few to me. Why weren't more individuals used? Did the authors make a power analysis to ascertain the appropriate sample size? Furthermore, although the authors had already studied the thermoregulatory behaviour of the Carniolan bees several times, I think they should have used the same number of bees as the other two subspecies. 

Response 5: Well, It is a misunderstanding. We used 16 chambers for the simultaneous registration of 16 worker bees: 6 Central European, 6 Caucasian and 4 Carniolan. However, we did not divide those groups in half. Sixteen Honeybees from three subspecies took part in the experiment in autumn, and the following 16 from those three subspecies in the spring. Altogether 32 bees were used. To avoid misunderstanding, we will modify the sentence: "We used 16 chambers with a temperature gradient to place individual worker bees.". We will write: "For each of two experimental turns - summer and autumn, we used 16 chambers with a temperature gradient to place individual worker bees of three studied subspecies."

Although we included not all of our experimental results in this manuscript, experiments took many days (much longer than those analysed here), making it impossible to use more bees during the same season with a limited number of thermal gradient chambers.

Honestly, we did not make a power analysis because all our other earlier experiments on the same sample size gave significant results. We always use samples similar in size.

Point 6: Is there a reference for the glycogen measurement method? Please, provide it. Same suggestion for the thermoregulation experiment.

Response 6: We added references for the glycogen measurement method: “Glycogen concentrations in the body of Honeybee worker bees affiliated with the studied subspecies were determined by absorption spectrophotometry using anthrone reagent [12-14]. The anthrone reagent was prepared on the day of the assay by dissolving 100 mg of anthrone in 50 ml of 95% sulfuric acid.”

For the thermoregulation experiment: “We conducted present studies according to the methodology developed and used in our previous research [16-21].".

Point 7: Line 120: Please, provide a description of the chambers used for the thermal preference recording. 

Response 7: We added to the manuscript:

“Fig. 1 presents the diagram of the experimental setup. To determine the temperature preferred by the bees, we placed them in an aluminium chamber (0.60 x 0.034 x 0.022 m), one of which was one end heated (by a built-in thermostat), and the other was cooled by a cryostat K21 E20 (GK Sondermaschinenbau GmbH – 103 Labortechnik Medingen). That generated a linear temperature gradient along the entire length of the chamber, ranging from 10-50°C. Due to temperature perception ability, the bees occupied the most advantageous (most appropriate) places in the thermal gradient chamber.”

We added a figure to the manuscript:

“Figure 1. Experimental set-up for the recording of thermoregulatory behaviour. A—thermal gradient chamber; B—fluid chambers; C—cryostat; D—thermostat; E—recording- keeper; F—computer; G—thermocouples; Linc—white light; Lred—red light and CAM—video camera.”

Point 8: Line 123: which food was supplied to bees?

Response 8: We added text in brackets to the manuscript:

“The researched bees had constant access to water and food (50% sucrose syrup).”

Point 9: The method of measuring thermal preference is not clearly explained. How many days did the experiment last? The position of the bees in the chambers was recorded for how long? What is the number of observations? Please explain in more detail. 

Response 9: We added to the manuscript:

“We recorded the location of the bees in the temperature gradient every 15 min for 10 s using a video camera connected to a computer, which made it possible to save movies immediately in AVI format on the computer's hard drive.”

At the end of the paragraph, we added:

“In the present paper, the determination of the thermal preference of the bees took three experimental days. After their completion, we kept continuing observations until the end of the life of the examined worker bees. The worked-out data on the survival time of bees and changes in their thermoregulatory behaviour during long-term (often several tens of days) experiments, we will present elsewhere.”

Point 10: Concerning the statistical analysis, did the authors consider models? Since the effect of multiple factors (season and subspecies) on a parameter is investigated, I think a better analysis could be achieved with a model. With a model, it can be better observed whether the effect of subspecies or bee season on the measured parameters is stronger.

Response 10: We considered the use of models, although analysis of variance seemed to us sufficient at this stage. We agree with the reviewer on the need to use models when dealing with multiple factors (season and species). We would like to do so, but it is impossible due to the short time allowed by the Journal to respond to the reviews and improve the manuscript.

Point 11: Data of average glycogen content have a very high standard deviation. The authors should consider and discuss this. 

Response 11: We agree with the Reviewer. In this case, the high values of the standard deviation were most likely due to the high variability of the studied bee populations, which is normal and often happens in the case of biological material.

Point 12: In figures 3 and 6, it should be specified in the caption what the white and black columns represent. 

Response 12: Corrected accordingly

Point 13: Line 214-216: I think this is a repetition of the introduction, which is not very functional for the discussion.

Point 14: Line 216-224: this whole paragraph should be shortened. 

Response 13 and 14: Accordingly, we removed a repetition for the introduction and shortened the whole paragraph.

“Overwintering ability is a crucial honeybee adaptation to unfavourable environmental conditions. It is derived from their different geographical origins and, as it can be assumed, behavioural and physiological responses, including changes in the chemical composition of body tissues.

In the present research, we studied the glycogen level depending on the season and subspecies affiliation. Additionally, we considered the worker  Honeybees' thermoregulatory behaviours. In those terms, we compared three Honeybee subspecies: Apis mellifera carnica P., Apis mellifera mellifera L. and Apis mellifera caucasica G.”

Point 15: In general, more bibliographical references could be included. Are there other studies to support the findings?

Response 15: We included more bibliographical references in the manuscript.

Round 2

Reviewer 1 Report

The authors responded comprehensively to questions and comments, and made almost all requested revisions. Where modifications were not possible, the authors provided an explanation. 

I think the manuscript has been improved and is acceptable for publication in its current form.