Peer review of: Bacteria evoke alarm behaviour in zebrafish (2019)



REVIEWERS' COMMENTS:  

Reviewer #2 (Remarks to the Author):  

In their revised manuscript, Dr Jesuthasan and colleagues report that bacteria contribute to the 
zebrafish alarm response, acting synergistically with a substance from the fish. They also present 
data on cell-in-cell formation by club cells, the source of alarm substance, suggesting this may 
contribute to their uptake of bacteria and mucus.  

The overall conclusion of the paper, that bacteria can evoke alarm behaviour, appears to be well 
supported and of interest to the field.  

However, the mechanistic conclusions on uptake of bacteria/mucus have changed significantly 
between the original and revised versions, and remain somewhat unclear.  

I was asked to provide a perspective on entosis. My view was that the original data did not support 
a role for this process and the manuscript has been revised accordingly. The reinterpreted data 
actually exclude entosis as a mechanism and the term has been removed from the working model 
(and cell cannibalism has been taken out of the title). While these changes do address the specific 
concerns raised, they do not provide a definitive alternative model.  

Major comments:

1) The data indicating that mucus/bacteria are internalised by club cells seems quite clear and the 
presentation of imaging data has been improved by the revisions. I am convinced that mucus and 
bacteria are present in club cells.  

2) However, the insights into the mechanism of uptake are still not well developed. In the original 
draft the authors gave significant weight to this part of their story. Cell cannibalism was included 
in their title and entosis in their working model, but in the revised version this mechanism has 
been excluded as relevant. This is quite a dramatic change in conclusions and there is limited new 
data to provide an alternative insight. Reference to both transcytosis and emperipolesis has been 
added, based on imaging observations. While some of the data would appear consistent with this, 
the relationships between these processes and mucus/bacteria uptake are not robustly quantified 
and not experimentally manipulated, leaving only a correlation. It is also unclear to me why 
phagocytosis has been excluded from the assortment of possible mechanisms?  

The question then is whether the broad conclusions of the paper are sufficient for publication 
without a more definitive mechanism, which is a matter for editorial judgment. In my view:  

a) If a mechanism for uptake is key to the paper, as it seemed in the original draft, the current 
data still require further development.  

b) If the paper stands without this detail, it would be advisable to reduce reference to uptake 
mechanisms further – for example, the negative data on entosis seem unnecessary (lines 85-99) 
and distract from the main thrust of the story.  

Minor points:  

1) Add graphs for some of the quantification mentioned only in the text e.g. in line 68, line 118.  

2) In some figures related to cell invasion (e.g. Fig 1d, 3n), the ‘internalised cell’ has no obvious 
DNA stain – this is not a convincing example of a ‘cell-in-cell’.  

3) Quantifying the association between cell invasion and WGA positivity would be helpful in 
building evidence for a mechanistic link (line 80).  

Reviewer #3 (Remarks to the Author):  

The authors followed all my comments and produced a very nice paper. I am convinced that the 
observations will set an agenda for possible future work in this area. Thomas Bosch, Kiel. (cid:3)