The Bacterium Pantoea ananatis Modifies Behavioral Responses to Sugar Solutions in Honeybees
Round 1
Reviewer 1 Report
The manuscript by Scheiner et al. entitled "The bacterium Pantoea ananatis modifies behavioral responses to sugar solutions in honeybees" is an important contribution to the knowledge on the drivers of insect feeding behavior. The ms presents original information, is well-written and well-structured, and the experiments and data analysis were carried out correctly. For these reasons, I do recommend its acceptance following addressing some minor corrections which I indicate in the attached revised version.
Minor corrections on english vocabulary need to be addressed, both in words and sentences. A reference is missing on the list which led to incorrect citations throughout the text and this needs to be corrected!
In a few experiments, it is not clear what was the solution used because it is stated differently in the manuscript (see attached file). There are also a few contradictory statements (see attached file).
Finally, I think that the manuscript will benefit from improving sections 2.4 and 2.5 and the corresponding sections 3.2 and 3.3. Particularly, in section 2.5 it is important to explain better the pre-test and its findings (which are not shown), but also the separation in two groups ("equal with respect to their sucrose responses") that are ignored in the results and the ommission on nectar and pollen foragers that are indicated in results. The discussion would also benefit from a more deep analysis of the behavioral findings comparatively to other animals (like the stated bumblebees, hummingbirds and slugs) and not just a listing of the other findings. The interesting findings that the indirect effects of bacteria (metabolites and changes on sugar concentration) seem not to influence honeybee behavior (as reported in other insect studies) should also be highlighted and discussed.
Best wishes
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
In this paper Ricarda Scheiner and collaborators investigated if pollen and nectar foragers of the honeybee Apis mellifera are able to detect P. ananatis bacteria in sugar solutions and if their responsiveness to sugar solutions would be affected by several bacterial. In doing this they also tested different types of sugar (namely sucrose, fructose, and glucose solutions). With a series of well-designed assays the authors showed that bees decreased their responsiveness to sugar solutions when these contained high concentrations of P. ananatis regardless of the nature of the sugar dissolved in water, the sugar concentration of the tested solutions and of whether bees collect nectar or pollen. Research question is well defined and relevant. The work is timely and original. The manuscript is clear and concise. The results are solid and statistically sound. In general methods are described with sufficient detail. I enjoyed reading the manuscript and I am really happy to say that I did not find any major issue. The work definitely worth publishing. I have only few minor points (listed below) that the authors may address prior to the publication of the manuscript.
MINOR POINTS
In general, I would invite the authors to a mild reconsideration of their interpretation of the results and the conclusions, particularly in relation to the ecological implications the results may have.
In the Synthesis and applications (lines 35-37) of the abstract the authors concluded that: “bees may not disperse P. ananatis bacteria in high densities during foraging trips and thus not contribute to the spreading of this potential pathogen”. Actually, I would say the opposite. Indeed, the results showed that
- Most of bees showed PER at low concentrations of P. ananatis in 30% sucrose/fructose/glucose solutions (lines 195-197).
- Bees are deterred only by very high bacterial density (yet, 50-60% of bees still responded to heavily contaminated sucrose (concentrations which likely exceeded those reported for nectar) (lines 201-202).
- Bees did not discriminate between pure sugar solutions and solutions containing bacteria at relatively low (D4) concentration (line 274-276).
- Bacterial concentration reported in nectar are about (3.1*104, line 287), which more or less is equal to that of D4.
Therefore, in my opinion (and perfectly in line with what the authors reported in the very last paragraph of the Conclusions of the paper, lines 309-311) these results support the idea that bees may introduce bacteria into the nectar and thus may affect nectar quality and plant fitness. What the authors reported when saying that bacteria in natural or agricultural ecosystems may affect the foraging behaviour of honeybees (lines 287 and lines 35-37) is not fully supported by the results.
Line 31: Authors may consider replacing “low bacterial concentrations” with “ecologically relevant concentrations”
Lines 189-190: is there any particular reason why you used SPSS rather than R just for this simple test?
Line 199 and elsewhere: Table 1 is missing or named in the legend as Table S1.
line 205: I was wondering how long the bacteria were kept in the sugar solution prior to testing. Is there any possibility that longer periods of time could lead to chemical changes in the solutions perceptible by bees?
Line 293: as discussed above “relevant densities of bacteria” is not fair, I would suggest replacing it with “high densities of bacteria”
Figure 1 and Figure 2a: reporting labels D1 D2 D3 D4 D5 next to the log bacterial density in the x-axis would help.
David Baracchi
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
It is an interesting study of the effect of high densities of P. ananatis bacteria on consumption sugar syrup by nectar and pollen collecting workers.
Th study is important for bee researchers and is therefore merit to publication.
General and specific comments and suggestions are enclosed to the manuscript (pdf). Some manuscript sections need to be rewritten. Materials and methods, Results and Discussion.
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
Scheiner et al studied the honeybee response to different concentration of bacterium P ananatis in sucrose solution and found that honebee response decreased with high conc of P ananatis.
It is very nice study with new ideas to know if pollinating insect could spread the different bacterium exist in floral nectar.
I have some comments that I might be bit harsh, but this will definitely improve the manuscript quality and presentation.
It has been said the author have used previously established method/protocol for the bee experiment i.e harnessed PER, I am not convinced this idea of harnessing. For example, when you give such harness any animals could react, and free flying bee is not different from that. Bee can count, perceive amount of nectar and can differentiate colors and so.. on.
When you harness the bee, the result will be skewed, so how do you present such a skewed result. Please clarify them.
What is the reason free flying bee have not tested instead? As I said free flying bee, behave differently while you test them. I am sure author know this and there might be several published too.
Some suggestions
Ln 62: author wrote P ananatis is useful bacteria but also said pollinators like bee do not disseminate it? Is not this a good thing if pollinator spread this bacterium which protects flowers and its reproductive organs against any other pathogen?? Any thoughts??
Method:
See above
Line 221-224: very confusing sentence, please revise it.
Figure 1D, it this a 30% or 3%, please correct it.
regards
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Authors responded adequately to my comments and suggestions and corrected previous manuscript version. The manuscript represents a valuable contribution to the field that merits publication in Insect
Author Response
Thank you for your suggestions.
Reviewer 4 Report
Author have addressed all the comments and clarified my questions. I would advise in the future please consider testing free-flying bee rather harnessing the bees even it is a standard method but someone has to change that ideas which do not give you the best result.
Regards
Author Response
Thank you for your suggestions.
