The manuscript by Doublet et al. addresses an interesting and important topic, namely the change of the honey bee virome due to the spread of Varroa from Asia to Europe and other parts of the world.
Varroa is not a vector of all bee viruses, but of some, but when Varroa transmits viruses to bee populations, the virome can change. However, the virome changes not only due to the transmitted viruses that adapt to the host, but the virome of a host can be affected by the ectoparasite that influences the host to facilitate parasitization - generally sucking blood, which in the case of honey bees is hemolymph.
In this very interesting paper, the authors studied the effects of Varroa infestation on honey bee viromes using historical samples (It should be clear throughout MS when and where samples were collected, especially for comparison with other studies.). The complete virome was not analyzed, but they analyzed the prevalence and titers of 14 viruses from honey bee samples collected in mite-free and mite-infested populations. A total of four independent geographic regions were analyzed. Analysis of 14 viruses is not analysis of virome, please clarify.
Importantly, when the authors claim to have investigated how the honey bee virome changes, they should have presented studies that investigated the virome. In general, virome refers to the assemblage of viruses that is often studied and described by metagenomic sequencing. Several studies have been done and therefore these studies should be presented and discussed. It should be of interest that the virome analyzed in Varroa naïve honey bee populations is different from that of honey bee populations that have been under the pressure of the mite for a long period of time. How does the virome of Varroa naïve bees differ from that of bees living in the area where the mite is ubiquitous (comparing the virome of bees from Australia and Europe)? For example, you can refer to the proposed difference in many different members of the Picornavirales due to Varroa. In addition to the best known DWV virus strains, the key difference should be the Acute Bee Paralysis virus (ABPV). Both DWV and ABPV are connected to the Varroa parasitisation.
I agree that the pathogens are transmitted by vectors, there is no doubt about that. Varroa is a mite and there is no mention of mite transmission. Although mosquito can be similar, it is not mite.
In addition, each parasite that transmits pathogens will affect host microbiota, but also its biochemical pathways are being changed. Change of the biochemistry of host is necessarily related to different pathogens and especially those that adapt to the host.
Line 70: varroa mites – Varroa mites (please check throughout the manuscript for similar typos)
Line 79: Varroa does not feed on fat body, it undoubtedly feeds on host blood similarly to ticks or another good example are members of Dermanyssidae (e.g. Dermanyssus gallinae). In the case of Varroa, the “host blood” is hemolymph. It is important to note that insects have an open circulatory system. In addition, when the ectoparasite sucks the host, it affects healing. The ectoparasite with the transmitted virus necessarily affects homeostasis and as a result autophagy is disrupted. Thus, when the mite tries to suck hemolymph, the cells of the host are also found in the gut of the mite. In the case of Varroa, if the mite prefers to suck near the fat body, the cells of the fat body may be ingested along with the hemolymph, especially if autophagy is disrupted. In addition, the mite feeds on different developmental stages, and especially during metamorphosis, the fat body undergoes remodeling.
Overall, it is misleading to say that the Varroa mite feeds on the honey bee's fat body, although it is in the title of a referenced paper.
Line 81: “prominent is deformed wing virus (DWV), the main driver“ – Is the DWV virus or the Varroa mite the main driver? I think the mite, because without the mite, the virus is not the driver of colony losses.
Lines 91-100 and the last paragraph in the background: to improve see above - virome studies, better explanation and link to virome change - the viral landscape can be presented
Line 114: “To determine the impact of varroa invasion on the honeybee virome“ – not clear, and virome was not analyzed
Line 116: considered to be varroa-free or varroa-infested
- Varroa free - not a real situation in the sites, although Varroa could be under control with miticides. Varroa symptoms observed/not obvious? Mites observed? Deformed bees observed? Please specify
Lines 120-121: “varroa-free areas“ – could you specify – it means that there were no mites as it was for example in Australia when researchers analyzed the virome ? Or were these areas without vorroosis symptoms? … Varroa was under control…
Line 128: Macula-like virus (BeeMLV, former Varroa destructor macula-like virus, or VdMLV). – it was initially identified analyzed especially in Varroa mites – should be better introduced / discussed … VdMLV was analyzed/detected and found previously high abundance in mites… Suggest search studies with the term “VdMLV and mite“
Together, DWV (the viral complex), ABPV (the viral complex), and VdMLV are obviously Varroa-transmitted viruses, it was known/suggested before your study.
Lines 211-213: it means that you analyzed DWV-B only UK and Canada? – not clear what means “surveyed ?”
Line 214: but, DWV-B is ubiquitous due to Varroa
Line 226: table S2 – Table S2
Lines 224-225: again, here and throughout the text, - what means „Varroa-free sites?“
Figure 1/2 – could correlation matrix be used?
Line 229-230: “in varroa infested populations, the highest viral titers were observed for DWV-A“
But DWV-B replaced the DWV-A due to Varroa mite or not? DWV-B is considered be more virulent
Figure 2 – „10 tested viruses,“ – but you analyzed 14 viruses
Figure 3 – Again, it is obvious that DWV-A correlates with Varroa, but studies say that DWV-B is associated with mite occurrence or not? How is it possible?

Line 268: “We hypothesized that varroa has affected the epidemiology of several bee viruses,“ ... However, this has already been hypothesized by a number of studies, and virome analyses have suggested a change in virome structure - possible replacement of viruses.
Line 272: the honeybee virome – virome analyzed different studies, and they should be discussed
Lines 272-273: “several other viruses have also become more prevalent, such as BQCV, CBPV, and SBV“ – due to Varroa or DWV?
Lines 274-276: “The emerging DWV-B, which has become increasingly dominant world-wide during the past decade [44], was not quite as prominent in the early 2010’s when these samples were collected,“ – This is where it becomes clear why DWV-B could not be identified in your samples - the time of sampling should be highlighted throughout the manuscript, including the abstract. Then it may become clear to the reader.
I suggest providing a schematic to make it clear what samples were used and when and where they were collected. Number of the above notes may not be written by the reviewer if it will be clear that you analyzed the samples before the proposed replacement of DWV-A by DWV-B? You can add the key studies to the scheme, when and where they reported the appearance of DWV-B.
Line 276: “these samples“ – I guess it means in your study
Lines278-280 “We found honeybees in mite-infested areas to carry significantly more virus species and a greater viral load than honeybees from varroa-free populations,“ - This may be true only for the set of viruses you are analyzing, but not if you were analyzing all viruses - the virome. In fact, the opposite is true.
Studies using virome analysis have shown that the number of virus species may be decreasing due to Varroa. See the comparison between the complete virome from Australia and Europe. At least many Picornavirales could disappear, but some different viruses, mainly DWV and ABPV complexes, could become dominant.
- Again, you did not analyze the whole virome, but "only" (compared to virome analysis) selected 14 viruses.

Line 283 “This variant was more prevalent“ – since when? In your samples? When they were collected?
Line 286 – “Experimental work“ – which ? – not clear
Lines 289-290: “The recently emerging variant DWV-B was seldomly detected in our historical samples.“ – Again, a clear indication of the sampling date would be helpful. And it should be clear which part of the discussion relates to your samples.

Lines 324-326: „The presence of varroa significantly changed viral titres of two other viruses: LSV-2, showing higher titres, and BSRV showing lower titres in honeybees from varroa-infested regions. LSV-2 has never been linked to varroa transmission and is rarely found in mites [73],“
- For the diversity of LSV viruses see virome analysis in honey bees – results in a study that compared different virome analyses – a phylogenetic tree shows the presence of LSV2 in some samples…
- Useful should be verify identifications in study/ies that analyzed virome of Varroa. Was LSV-2 found in Varroa virome?
I suggest to verify the correlation of BeeMLV/VdMLV with the presence of Varroa, since this virus has been previously identified in the mite at high titers, and it has also been identified high-abundance at the proteome level in the mites, supporting the high abundance in mites. Relevant discussion may be useful. It may explain the following „Lines 194-195: Note that models were not run for ALPV, BeeMLV, DWV-B and SBPV as too few samples were positive.“