REVIEW OF THE MANUSCRIPT:
“Marine transmissible cancer navigates urbanised waters, threatening to spillover”

GENERAL COMMENTS

The manuscript from Hammel et al. investigates the prevalence of one mussel BTN lineage in an area of France that was intensively sampled (76 locations) and where two mussel species cohabit. Genetic analyses were carried out to diagnose and inquire about the genetic background of the affected individuals. Finally, statistical analysis of environmental variables was performed to through some light on the epidemiology/propagation of these mussel contagious cancer (MtrBTN2). I found this study very interesting and provides data supporting the impact of maritime routes in the propagation of mussel contagious cancers plus the role of several factors (density, pollution…) in ports favoring these sites as epidemiological hubs. I have a number of questions and comments:

• Authors suggest preventing biofouling on docks and ships’ hulls to limit the spread of these cancers, I miss the discussion of some biofouling alternatives.

• Regarding environmental data (in particular, temperature and salinity), why was it preferred to use public databases instead of recording them during the sampling? How accurate/close to the sampling location are the measurements of the databases recorded?

• New primers of mtCOI and EF1a genes were designed for this study, as these markers have previously been used in several previous studies, how are these new primers improving the annealing/amplification of those sites?

• In the pooled screening step (EF: 9/127 – mtCR: 14/127 – both: 2/127), it is clear that both markers would be the desirable result, it is clear why mtCR positives had low copy numbers and that could lead to not detect the EF but I need more explanation on why 9 samples (36% of positives -9/25) only amplified the nuclear marker that is expected to be in low copy number compared to the mitochondrial copy number.

• Authors acknowledge that the sample sizes per site may be too small (n=20) to detect BTN, which factors were taken into consideration to decide the sample size for the sites in this study? Was the distance between the collection points taken into account for the analysis?

MINOR COMMENTS
-Line 42: speaking in general about BTNs, I suggest removing the word sessile as it is true for mussels but not for cockles or clams that can move (although with very limited movement)

Congratulations on this study!