Reviewers' comments: The present paper focuses the characterization of nucleic acid hydrolysis by protease from snakehead stomach and their constituents – pepsins SP1 and SP2. Nucleic acids such as λDNA and DNA from salmon sperm were used as potential substrates for snakehead stomach protease. Basic properties of pepsin-dependent NAs hydrolysis, such as pH-, temperature-, and time-dependences were evaluated. NA-hydrolyzing capacity of total (non-separated) stomach enzyme from snakehead and bovine as well as banded grouper pepsins was compared. This paper reports on the nucleic acid hydrolyzing properties of native acid proteases of fish. Strong evidence of λDNA hydrolysis in the presence of proteolytically active fish pepsins was obtained; but paper rise some issues according to data interpretation.

Specific comments to the manuscript: Introduction - Line 28 – describing the products of NA hydrolysis a sequence “oligonucleotide – mononucleotide – nucleoside – free base” seems to be more correct. - Line 42 – a statement that “…fish pepsins possessed the ability to digest NAs, but bovine stomach enzymes did not…” is to some extent argue because of the reason indicated in the comments to Result and Discussion section (see below). Materials and Methods - Page 4, lines 25, 33, and 47 refer to “Section 2.5”, although the section does not contain numbered subheadings. Results and discussion - Studying pH-dependence of NA digestion (see Fig. 2 A, B) authors obtained predictable results on NA acid hydrolysis (at pH range from 2.0 to 3.0) independent on the presence or absence of SP2 protease. The same result (complete NA hydrolysis) was obtained for bovine stomach enzyme (Fig. 7, B). Why pH 2.5 for banded grouper pepsins was used to compare fish and bovine protease NA-hydrolyzing capacity (Fig. 7, A vs B)? This pH value does not allow resolving a problem to distinguish non-enzymatic acid hydrolysis and pepsin-dependent hydrolysis as these processes have similar pH optimum (2.5) according to the data on Fig. 2 and Supplement Fig. S1, A. - The authors have not supposed any possible mechanism of DNA-protease interaction allowing protease-dependent NA hydrolysis. What bonds in NA structure could be attacked by proteases instead of amide (or peptide) bonds in polypeptide chain (their convenient specificity)? For my instance, it should be discussed. References - Reference McAllan (1980) is incomplete (see paper title) Table and Figure Legends (page 12) - Fig. 4 (A), lines 24-27 – factual errors on figure legend (but not on a figure): Lane 1 – should be indicated as “without treatment” (instead of “was treated”), lane 2 – as “with pre-treatment” (instead of “without pre-treatment”) - Fig. 7 – no “M: protein marker” lane indicated on figure 7 legend (line 48) is presented on corresponding figure; - Fig. 7 (A) – the difference of 1, 3, and 5 lanes (as well as of 2, 4, and 6 lanes) is not indicated on Figure legend, apparently three grouper pepsins (I, II, and III) were loaded on corresponding wells; this information maybe important and should be included. Referee conclusion. To sum up, a revised manuscript by Yu Liu et al. that effectively addresses the issues raised can be accepted for publication in Fish Physiology and Biochemistry journal.