Successful Indoor Mass Storage of Honeybee Queens (Apis mellifera) during Winter
Round 1
Reviewer 1 Report
In this work titled “ Successful indoor mass storage of honeybee queens (Apis mellifera) during winter” Andrée Rousseau and Pierre Giovenazzo investigated the survival of young and mated queens stored massively in queenless colonies over almost one year (8 months). These queen banks were kept in controlled rooms at different temperatures, either above or below cluster formations. In evaluating the impact of this indoor mass storage on honeybee queens they evaluated queen survival and both queen fertility and fecundity. With a series of simple analyses the authors showed that the long-term indoor mass storage of mated queens during winter could be achieved with success when queen banks are stored above cluster temperature at 16 °C. moreover they also showed no significative impact of long-term indoor storage on sperm viability or abdomen width of queens, although stored queens incurred a length reduction of their abdomen following wintering compared to control queens. Therefore the authors concluded that their proposed method to storage queens in captivity under properly controlled conditions is a potential approach to fulfil the high demand for queens in early spring. Research question is well defined and quite relevant. The work is timely and original. The manuscript is quite clear and concise. The results are solid and clear. In general methods are described with sufficient detail. I enjoyed reading the manuscript and I am I did not find any major issue. I have only few minor points (listed below) that the authors may address prior to the publication of the manuscript.
MINOR POINTS
In general, even if I am not a native English speaker, I think that the English should be improved a bit, especially in the discussion. Several typos are present throughout the entire manuscript.
In the Material and Methods the authors stated that 630 queens were produced by three local queen breeders. Do the authors have any idea from how many different nuclei? To what extent the queens differed genetically?
The authors also explained that queens were raised using the grafting technique and harvested once mated. Does it mean that the queens were mated naturally? If this is the case, please make it explicit. If not, please specify with how many males they were mated.
Fifteen queen banks were distributed into three groups (N=5 queen banks/group) and assigned to three different controlled rooms: 6 °C ± 1 °C; 11°C ± 1 °C; 16°C ± 1 °C. Control queens in standard colonies were placed in an environmentally controlled room at 6 °C ± 1 °C. Is there any reason why the authors did not place control colonies also at 11°C ± 1 °C and 16°C ± 1 °C. In my opinion this would have been a more comprehensive control.
In the Results section the authors reported that “queens in control colonies had a greater body weight than queens banked 225 at different temperatures (Figure 6) (χ² (4.55) = 19.668, P = 0.0006)”. Did the queen have more developed ovaries and/or more eggs? They briefly discussed this point in the discussion section, but I wonder whether they had the change to check the abdomen of at least some of the queens.
In the paragraph 3.3 the authors state that most queens from 241 various queen banks were fertile: laying eggs and with worker progeny (53%, 71% and 242 68% respectively). Are these percentages calculated as percentage of the total number of initial bees or of the survival queen at 7-10 days? Please specify it in the text. This information can be calculated from the table, but I think it might be specified in the text as well to help the readers.
In the discussion section (Desai and Currie 2016) should be in number. Please amend it
Author Response
Author's Reply to the Review Report (Reviewer 1)
Minor points
In general, even if I am not a native English speaker, I think that the English should be improved a bit, especially in the discussion. Several typos are present throughout the entire manuscript.
- Typos have been corrected throughout manuscript and a second round of proofing was done.
In the Material and Methods the authors stated that 630 queens were produced by three local queen breeders. Do the authors have any idea from how many different nuclei? To what extent the queens differed genetically?
- Information added line 77.
The authors also explained that queens were raised using the grafting technique and harvested once mated. Does it mean that the queens were mated naturally? If this is the case, please make it explicit. If not, please specify with how many males they were mated.
- Information added line 78.
Fifteen queen banks were distributed into three groups (N=5 queen banks/group) and assigned to three different controlled rooms: 6 °C ± 1 °C; 11°C ± 1 °C; 16°C ± 1 °C. Control queens in standard colonies were placed in an environmentally controlled room at 6 °C ± 1 °C. Is there any reason why the authors did not place control colonies also at 11°C ± 1 °C and 16°C ± 1 °C. In my opinion this would have been a more comprehensive control.
- Our control group represents the normal method of wintering colonies with a solo queen, we have considered this a control group of wintered queens. But, in light of the reviewer’s comment, we are anticipating new research that aim to revise of existing wintering recommendations.
In the Results section the authors reported that “queens in control colonies had a greater body weight than queens banked 225 at different temperatures (Figure 6) (χ² (4.55) = 19.668, P = 0.0006)”. Did the queen have more developed ovaries and/or more eggs? They briefly discussed this point in the discussion section, but I wonder whether they had the change to check the abdomen of at least some of the queens.
- We did not do ovary measure, this is now being tested 2020-2021.
- But a follow up of these queens in colonies during summer indicate normal ovary function.
In the paragraph 3.3 the authors state that most queens from 241 various queen banks were fertile: laying eggs and with worker progeny (53%, 71% and 242 68% respectively). Are these percentages calculated as percentage of the total number of initial bees or of the survival queen at 7-10 days? Please specify it in the text. This information can be calculated from the table, but I think it might be specified in the text as well to help the readers.
- Information added line 242.
In the discussion section (Desai and Currie 2016) should be in number. Please amend it
- See line 283.
Reviewer 2 Report
Dear Authors,
the paper presents very promising results about the long-term overwintering of honeybee queens inside queen-banks. The temperature ranges tested could be very useful also for novel studies and trials about overwintering queen banks in other climatic contexts. Moreover, this practice can provide huge benefits to the beekeeping sector from the productive, sanitary and species-protection points of view.
Please find attached only few comments that could help readers (and beekeepers) in understanding and applying this technique.
Comments for author File: 
Comments.pdf
Author Response
Author's Reply to the Review Report (Reviewer 2)
From reviewer’s pdf
- Line 79: typo corrected
- Line 109: colonies were not fed any sucrose after fall feeding 40L. In April all banking colonies had leftover sucrose reserves from fall feeding. Information is added line 111 and line 214
- Line 115: the origin of the breeder was not taken into account for the stats.
- Line 147: information added line 150
- Line 232: The figure will be given to editor in high resolution
Reviewer 3 Report
The current study is about queen bank environmental condition impact on queen survival and fitness. The authors used a huge number of colony to test their hypothesis. The very high number of queens highly contributes to the strength of their study. By testing the impact of different temperature they were able to suggest the best temperature for indoor raising conditions to increase queen survival in Canada.
The paper is well written.
I have few questions.
First the authors didn’t explain how the queens were mated. Were they naturally mated, at which step of their protocol. As the authors did count spermatozoid and their viability, they should just explain how they proceed for this step.
My second main point is the authors highly the impact of mass storage without really measuring it. Could they define clearly what they considered as mass storage? It’s not intuitive and their results didn’t show any measurement of such variable.
The paper also addresses only literature on honeybees. Literature is also available regarding the impact of mass storage on other Hymenoptera. (such as Wasps, publication Hunt).
I was not able to read the graphic legend of the figure 6
Comments for author File: Comments.pdf
Author Response
Author's Reply to the Review Report (Reviewer 3)
From report
First the authors didn’t explain how the queens were mated. Were they naturally mated, at which step of their protocol. As the authors did count spermatozoid and their viability, they should just explain how they proceed for this step.
- Information added line 133
My second main point is the authors highly the impact of mass storage without really measuring it. Could they define clearly what they considered as mass storage? It’s not intuitive and their results didn’t show any measurement of such variable.
- This is a curious question… Nevertheless I hope this answer is ok: Our research compares survival, fertility and fecundity of winter banks of 40 queens with normal one queen per colony wintering.
The paper also addresses only literature on honeybees. Literature is also available regarding the impact of mass storage on other Hymenoptera. (such as Wasps, publication Hunt).
- Wasps are quite different during wintering as only the queens overwinter solo. Honeybees overwinter as colonies, one queen and many workers.
I was not able to read the graphic legend of the figure 6
- After verification, the legend is ok
From PDF
Line 79: typo error is corrected
Line 84: these 30 colonies were grouped in order to create 15 very strong queenless colonies. The queen banks were placed in these colonies.
Line 94: 600 queens = 15 queen banks each with 40 queens. The 30 other queens were used to create 20 control solo queens each in a colony (ref line 110). The extra queens were used for Time 0 evaluation.
Reviewer 4 Report
The paper explores the research subject of an indoor mass storage of mated honeybee queens with an aim of their effective successful utilization in the following spring.
The authors have examined survival, fertility and fecundity of young, mated queens stored massively in queenless colonies by performing experiments where the queens have been kept at three different temperatures in environmentally controlled rooms.
The findings have shown a significantly higher survival of mated queens when queen banks are stored above cluster temperature at 16 °C.
I appreciate that the data are coming from the original research conducted in Canada employing the queens produced by three local queen breeders and a research center, where all experiments were conducted.
The research was funded by Quebec’s Ministère de l’Agriculture, and by the Natural Sciences and Engineering Research Council of Canada.
The manuscript is well written and is comprehensive. I find the issue relevant. The paper is well done and I haven't remarks.
Author Response
Author's Reply to the Review Report (Reviewer 4)
From report
The manuscript is well written and is comprehensive. I find the issue relevant. The paper is well done and I haven't remarks
- No reply needed
