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Laboratory of Insect Genetics and Biosciences (IGB) Dept. Biology – University of Modena and Reggio Emilia Via Campi 213/D, 41125 Modena (Italy) Tel. +39-059.2055544 – Fax +39-059-2055548 Homepage: http://www.igblab.it Modena, 03.08.2015 Dear Professors Xu Guo and Vladimir Teif, I am sending you herewith enclosed the revised version of the review entitled “Cytosine methylation in insects: new routes for the comprehension of insect complexity” which we would like to submit for publication to the special issue “Chromatin and Epigenetics” of AIMS Biophysics. In the present version of the review, we modified different sections according to the referee’s comments in order to have a more focussed review. For some comments, we are not in agreement with referees so we explained why some changes are not due in our opinion. In particular, according to REFEREE 1: Mandrioli and Manicardi review the existing evidence that epigenetics may play a role in insects. Specifically, in phenotypic differences between clonal aphids and in the development of eusociality in hymenopterans. Although the idea is interesting and a review on the topic may be timely, the claims made are not supported by the existing experiments. First, the authors claim that differences at phenotypes between clonal aphids are due to epigenetic changes triggered by the environment. However, the papers revised show that there is methylation in aphid genomes, and that experimental manipulation of methylation patterns alter the phenotypic outcome. There is no evidence that these changes are triggered by environmental changes. I am 1 not suggesting that this does not happen, but the literature reviewed does not show, in my opinion, support for such a statement. R: Changes in the aphid morphology (including for instance colour and wing presence/absence) are triggered by the environment, according to a large set of data. As assessed by Dombrovsky et al., changes in colour and/or wing presence is due to different methylation levels (as also assessed using DNMT inhibitors) so that we agree with Dombrovsky et al that aphid epigenetic changes have been triggered by the environment. Even if we agree that further molecular evidence could make our statement more convincing, we think that a support is already present in the currently available data. Second, the authors nicely review the evidence available about methylation in bees. From the literature it is obvious that methylation happens, and that different castes have a different methylation profile. However, the text goes too far on suggesting that the origin of eusociality is explained by the acquisition of epigenetic mechanisms. There are many 'traditional genetics' explanations which, so far, have support from models and experiments (see for instance the works by W. Hamilton, G. Williams and M. Nowack, among many others). Any discussion about the origin of eusociality should include these works in an appropriate context. R: we just partially agree, since previous studies discussed the evolutionary mechanisms that make possible the success and spread of eusociality in term of kin/group selection, inclusive fitness theory and so on from an evolutionary point of view. This means that they have not focused on mechanisms that “made” castes, but on the reason of the success of these superorganisms. Classical genetics helped therefore to understand the effect of natural selection on eusociality, but they did not explain the molecular machinery at the basis of castes that are generally described as environmentally-based in eusocial insects. In our paper we refer to the mechanisms that make possible to have castes at a morphological level. However, we changed different sentences in order to avoid any misinterpretation of our suggestion. 2 Minor Comments: What West-Eberhard and Waddington would have called epigenetics is different to what we understand today as epigenetics. Their works should be, therefore, interpreted within their context. R: we revised our text and deleted the figures in order to avoid a misinterpretation of the terms used by West-Eberhard and Waddington in respect to the current meaning of the same term. Despite the author's claim, Molecular Biology's Central Dogma has no problems with Epigenetics. Also, epigenetics is a main theme in mainstream genetics. R: We have no doubt that epigenetics is a main theme in modern genetics and we think that epigenetics (that include different mechanisms, such as noncoding RNA) is not properly in agreement with the central dogma, since microRNA can transmit information relevant for the phenotype to DNA, whereas in the central dogma information are always transmitted from DNA to RNA. We modified our sentence in order to make clearer our idea. When the authors refer to epigenetics in Drosophila, what are they referring to? (There is no DNA methylation in Drosophila, for instance, so they may write about a different mechanism). R: Several papers reported the presence of DNA methylation in Drosophila during the fly development at non CpG target. Furthermore, even if at low level, the presence of DNA methylation has been detected also in adults. In particular, in adults several papers unequivocally confirmed DNA methylation at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. We also reviewed data on flies with Field, Prantera and Lyko in 2004. Figures are not referred in the main text. 3 R: Figures have been deleted According to REFEREE 2’ comments: 1. The title of the review is “Epigenetics in insects: new routes for the comprehension of insect complexity”, but the review is about DNA methylation, which is just a portion of all know epigenetic marks. Title should be changed to focus on DNA methylation only (e.g. “DNA methylation in insects: new routes for the comprehension of insect complexity”), or alternatively the authors should add consistent amount of information about additional epigenetics mechanisms (for example histone marks studies performed in Drosophila, such as PMID:21179089) R: it is true, we revised our title 2. In the introduction, DNA methylation function is described in promoters and genes, but does not mention additional genetic features. In particular, methylation at TEs is directly involved in silencing, while the function of body methylation is not clear, and only in some cases correlate with increase expression. At least in plants, the removal of all body methylation (occurring in met1 mutant and epiRILs) has no clear effect on gene expression (PMID:19390088). R: we modified our introduction making a clear statement about the role in transposon silencing 3. The review focuses on two main models (bees and aphids), and is not mentioning the most studied insect, Drosophila melanogaster. Recent studies identified the presence in low amounts of DNA methylation in Drosophila melanogaster (PMID:24640988; PMID:24558263; PMID:25936838), and this review about DNA methylation (or epigenetics) in insects should at least mention these studies. 4 R: It is true, but data on DNA methylation on flies has been already frequently revised and discussed. So we decide to focus on less popular experimental models. However, we better described our choice at the end of the introduction 4. The review only mentions about 5 methylcytosine (5mC). At least in the introduction, the authors should discuss about additional DNA modifications, such as 5-hydroxymethylcytosine (5hmC) (PMID:25100549) and N(6)methyladenine (6mA) (PMID:25936838) (both seem to be present in insects) R: The presence of 5hmc is described in insects but it is not clear the function so that we decided to focus on 5mC and the changed several sentences in order to explain our focus. 5. The authors claim “5mC is found almost entirely within CpG dinucleotides in the DNA of mammalian somatic cells”. A recent paper showed the existence of non-CG methylation in mammalian somatic cells (PMID:26030523). In fact, non-CG methylation in mammals seems to be more informative of gene expression than CG methylation. R: It is true that DNA methylation has been detected also in non-CpG target but as a whole at present most of the DNA methylation is at CpG considering mammals, so that we modified our text suggesting that most and not entirely the DNA methylation is at CpG. 6. Recently, it was shown that differences in conservation rates between different genetic features can bias the detection of the methylation levels (PMID:26024968). The authors should mention about this results and how to compare the methylation levels when dealing with different conservation rates. R: This is true and this is why we decided to compare just the amount of 5mC to distinguish highly and poorly methylated genomes (with insects in the second category) without focussing on conservation of methylated sites. This analysis could be very difficult in aphids and honey bees since the different morphs/castes differ in the methylated sites. 5 7. According to the review, morphological traits can be explained only by epigenetics. TFs are known to influence morphological traits and even induce pluripotency. The review should at least mention about TFs and their control of gene regulation (PMID:23584020). R: it is true, so that we add some sentences explaining why we put such an emphasis on epigenetics in place of other elements involved in transcription control. All authors agreed that the revised manuscript will be considered to be resubmitted in AIMS Press journal in Open Access Format, and the submission has not been published in another journal. Looking forward to hearing from you, Sincerely yours, (Prof. Mauro Mandrioli) 6