Review History

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  • The initial submission of this article was received on May 23rd, 2017 and was peer-reviewed by 2 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on June 8th, 2017.
  • The first revision was submitted on August 14th, 2017 and was reviewed by 1 reviewer and the Academic Editor.
  • The article was Accepted by the Academic Editor on August 21st, 2017.

Version 0.2 (accepted)

· · Academic Editor


Good news to start off your week! I'm happy to pass along a decision to Accept your manuscript for publication in PeerJ. Congratulations!

Reviewer 1 ·

Basic reporting

This is a revised manuscript by Lonsdale et al. investigated monoallelic DNA methylation and expression in the bumblebee. Overall I find the manuscript much improved and my initial concerns have now been satisfied.

The language, article structure and discussion all read well and the manuscript is well written and easy to read. Appropriate citations are used. The discussion of genomic imprinting has now been toned down as appropriate.

Experimental design

The additional figures and explanations in the text greatly add to the manuscript and increase the clarity of what was done and what conclusions can be made from the data.

Validity of the findings

Speculative results have now been put in context and caveats are now listed as appropriate.

Comments for the author

A very nicely revised manuscript which I agree is a much improved version of the original submission. I look forward to seeing it in print!

Version 0.1 (original submission)

· · Academic Editor

Major Revisions

Please see the reviewer comments. Both are quite positive, but also suggest some critical issues, particularly with regard to the figures and suggest some additional data that could be presented.

I encourage you to address these comments to the best of your ability and look forward to a resubmission.

Reviewer 1 ·

Basic reporting

The manuscript is well written with clear and professional english used throughout. Literature is appropriately referenced and sufficient context to the paper is given.

The figures are quite disappointing and do not add to the manuscript. In the main manuscript, only one figure is shown (gene ontology) and this is not discussed adequately in the text. What is the biological significance of these enrichment categories? What conclusions do the authors derive from this? The supplemental figure is not well described and confusing. Should there be dots in figure b? What does 'tagwise' mean?

I would really like to see a gene browser view of one/some of the genes identified in this study, showing the distribution of reads from the three sequencing methods around the gene. This would really help give a sense of the data to the reader and help visualise the results and concepts trying to be conveyed. Much more data can be presented in the figures which currently are very limited.

Table 2 is missing.

line 188 has incomplete information: "x, x"

Experimental design

The manuscript is within the scope of PeerJ. The research question is well defined, relevant and meaningful and worth investigating.

The methods are generally well described with the exception of the bioinformatic analysis of the methylation data which could benefit from additional explanation. Additionally, regarding the gene ontology analysis, could the authors clarify what background gene set was used to perform the analysis.

I am unclear whether the combination of MRE-seq and MeDip is suitable to identify allele-specific methylation given the inherent biases given by each method. Could the authors provide supporting information or validated bioinformatic methods to confirm that the results seen are not an artefact seen by the different enrichment methods? Alternatively, would it be possible to validate one or more of the loci by bisulfite-PCR based methods?

Validity of the findings

While the hypothesis is interesting and deserves investigation, the study does not appear to be developed but remains at a preliminary and unfinished stage. Unfortunately the manuscript and figures reflect this.

There is clear speculation regarding the role of genomic imprinting in this context. While this has been identified as speculative, there is absolutely no evidence suggesting that this is the case. Of the validated expression patterns, none showed a clear bias of one allele in one individual and the reciprocal allele in another which would be supportive (but not conclusive) of imprinting. It is more likely that the results are due to underlying genomic variations which are affecting the expression of the allele in cis. I would suggest to tone down the claims to genomic imprinting, especially in the abstract.

Comments for the author

Great hypothesis and I was really caught and intrigued by the end of the introduction. Unfortunately I find that the results are minimal and left me wanting a fuller and deeper description of what is happening in this very interesting system.

Reviewer 2 ·

Basic reporting

The article was generally well written. The ideas were clear. The questions were put into appropriate scientific context.

Experimental design

See 'General Comments'.

Validity of the findings

See 'General Comments'.

Comments for the author

Review of “Allele specific expression and methylation in the bumblebee, Bombus terrestris” by Lonsdale et al, PeerJ ms #18128.

The authors investigate the relationship between allele-specific expression (ASE) and DNA methylation in the bumblebee. They find some evidence of a link between ASE and gene methylation. They interpret their results in light of the importance of imprinting and control of gene regulation in insects.

The study of ASE is of considerable importance to the scientific community. The subject is of particular interest to those studying social systems, because of the possible importance of gene imprinting in these cases. Thus I think the general subject area is worthy of study.

The authors did find some evidence for a relationship between methylation and ASE in their samples. However, I was somewhat skeptical of how strong this finding was, given the methods used. That is, bisulfite-sequencing combined with RNA-Seq is the gold standard for studying methylation-expression relationships. But the methods used here, and the use of data from a variety of studies, were not as strong. Nevertheless, I still felt the study was worthy given the context of the study and journal. But I would like to see the authors add a few more caveats to their Discussion regarding possible issues with their analyses.


The Introduction is well written and reflects the state of the field. I particularly like the fact that the authors accurately discuss the lack of clarity and consensus surrounding the field of epigenetics in social insects.

82 It looks like the authors have only one ‘replicate’ of their laboratory work (i.e., they only analyzed one bee). This is unfortunate as replication of methylation analyses is now the standard of work. I am sympathetic to the difficulty in being able to carry out these studies. But the use of only one replicate weakens the study and thus the results here must be considered with some caution.

90 I’m not familiar with MRE-Seq and was a little confused about what was being derived from this analysis. The authors state that “MRE-seq enriches for unmethylated cytosines” and so I expected that this would mean sequences obtained from the MRE-Seq analyses would represent unmethylated sections of DNA. But later (106) the authors state that “We searched for genes that were monoallelically methylated (present in both MeDip-seq and MRE-seq libraries)” which suggests that MRE-seq procedure is actually enriching for methylated, rather than unmethylated, DNA. Can the authors be clearer about this?

106 Following up on the query above, the authors state that “We searched for genes that were monoallelically methylated (present in both MeDip-seq and MRE-seq libraries)”. But does this mean that they discovered one allele in the MeDip-seq library (the putatively ‘methylated’ allele) and the other allele in the MRE-seq library (the putatively unmethylated allele)? Is that how they identified monoallelically methylated alleles? Please be clear.

It seems that at least some of the RNA-Seq analyses were not conducted on the same bees (or even the same tissues?) as was used for the MeDIP and MRE analyses. Is this correct? If so, this should be acknowledged and discussed. It is possible that the link between methylation and ASE may be tissue specific which might confound the authors' analyses.

In general, the authors should discuss from which tissues (brains? Whole bodies?) the various samples were obtained. This could be quite important if they are trying to link methylation to expression. Did all studies analyze the same tissues? If not, this needs to be discussed.

Detecting heterozygosity isn’t trivial. For example, a single sequencing error might give the appearance of heterozygosity in the sample even if it isn’t real. In principle, there are ways of handling this. You might expect ‘true’ heterozygotes to show 50% of each sequencing allele. The authors should discuss this a bit and explain how they differentiated true heterozygotes for artifacts.

132 The authors provide helpful information on the ‘coverage’ of their libraries. This is certainly useful. But it does raise the question of how allele specific expression was detected and how accurate it is. This is not a trivial issue, as the detection and confidence that an allele is showing allele-specific expression depends on the number of reads that come from that allele, which may depend on the length of the gene and other factors. These issues have been considered before and they deserve a bit more consideration and discussion here.

194 Much of the analyses the authors undertake relies on the counts of sequences obtained in their libraries. The authors presumably excluded genes that had low count numbers (e.g., a gene have only a single count in the RNA-Seq library means that it would always be viewed as showing ASE). But knowing the count numbers for each gene in each of their libraries would provide insight into the robustness of the results. Could this information be provided in Sup Table 1?

I was little confused as to whether figure 1 had some kind of meaning I wasn’t getting. Is this essentially just a list of GO terms associated with genes displaying allele specific expression? Or does the structure of the figure have any meaning? For example, does the fact that certain terms are at the top of the box have any meaning? Or does the fact that some terms are next to each other (e.g., hatching behavior and phosphate-containing compound) have any meaning beyond what is conveyed by the colors. If this figure actually does not convey any meaning (as a figure) the information may better be provided in a table.

The Discussion is generally well written and acknowledges the complexities of interpretation of methylation information in insects.

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