Download Laboratory of Insect Genetics and Biosciences (IGB) Dept. Biology

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
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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.
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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.
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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.
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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.
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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)
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