The Marine Sponge Petrosia ficiformis Harbors Different Cyanobacteria Strains with Potential Biotechnological Application

Round 1

Reviewer 1 Report

The authors isolated cyanobacteria strains from the marine sponge Petrosia ficiformis and characterized their morphology and phylogenetic relationship. They also investigated the biological activities of the cyanobacterial extracts on mammalian cells. I feel that this manuscript is well written and could be published in JMSE journal. The manuscript could be improved with the following points.

Major points.

1,  The authors stated that they purified cyanobacteria strains from P. ficiformis by streaking. However, I could see some particles that look rod-shaped bacteria and do not show chlorophyll fluorescent in Fig 1a, b, c, and e. 

The Authors stated that cyanobacteria strains used in this study are axenic, but no evidences were shown. Their PCR primers for rRNA sequencing were designed specifically for cyanobacteria, so it cannot detect the contamination. Generally, isolation of axenic culture is not an easy task, especially for filamentous strains. So I strongly recommend authors including the DAPI images in Fig 1. Otherwise, the authors should be less conclusive about the axenicity of these strains.

2, The sequence length of the 16S_ITAC_102 (~600bp) is shorter than the other strains, which could result in the lower bootstrap value of this strain compared with other strain in Fig. 2. Therefore, the sequencing and phylogenetic analysis of ITAC_102 strain should be performed using ~900bp amplicon.

3, I would suggest the authors discussing and/or comparing the effect of the cyanobacterial extracts on the MTT or morphological assay with other studies. Are there any other studies performing the same assay using cyanobacteria extracts on mammalian cells?

Minor points.

1, L214, the authors should describe the number of cells used for calculation of the average and standard error (or standard deviation) of the cell size.

2, Please convert the uppercase letters in the title of references to lowercase. 

For example,

"Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria" (Ref. 2)

should be written as

"Natural product biosynthetic diversity and comparative genomics of the cyanobacteria".

Please check all the titles in the reference.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript, “The marine sponge Petrosia ficiformis harbours different cyanobacteria strains with potential biotechnological application” describes the microorganisms isolated from a Mediterranean sponge that the authors have pursued for interesting chemistry, pharmacology, and biology. The organisms have been studied by a polyphasic approach to specifically identify them. This is especially important because some of the strains have been shown to produce natural product chemistry or macromolecular constituents with pharmacological value, such as killing cancer cells in vitro, modulating cellular morphology, and altering mitochondrial activity. I am recommending that this work be accepted after minor corrections, with some examples of why that follow:

There are some minor grammatical errors throughout the manuscript. I am listing several early examples here to facilitate corrections, but the list is not comprehensive and the document should be carefully revised for grammar specifically.

  Line 26 “…with some of the cyanobacterial strains showing toxic activity…” – the strains did not show the activity, rather the aqueous cell supernatant did.

  Line 52 “…association with sponge…” – should be “…association with the sponge…”

  Line 55 “…defense throughout the production…” – should be “…defense through the production…”

  Line 58 “…cyanobacteria resulted responsible for lysis…” – should be “…cyanobacteria aqueous cell supernatant was responsible…”

Some aspects of the methodology or presentation are not clear and should be improved.

  Line 76-78 describes the large scale cultivation of the cyanobacteria but fails to mention how long these (slow-multiplying) organisms were grown for. The information on Line 134 is that it was 2 months. I believe this should be included at line 78.

  Line 141 “The supernatants, representing the aqueous extracts, were recovered…” – this is wrong, since there was not an extraction protocol or an aqueous solvent utilized. This should be corrected as “The supernatants were recovered…” and the “extract” should be replaced as “aqueous supernatant” throughout the document.

  Lines 121-132 describes molecular analysis methods for producing the phylogenetic trees shown in Figure 2, however it is unclear why these were conducted separately instead of making one tree, and none of them has included an outgroup. This needs to be explained and/or corrected.

  Line 237-240 (style rather than methodology) Furthermore, the Figure 2 caption appears on Page 7 although the figure appears on Page 6, which only serves to confuse the reader.

  Line 207 (Figure 1 style rather than methodology) The scale bars on the bottom row should be propagated across all panels, as they have been appropriately already in Figures 4 and 5.

  Line 268-293 describes the effects of the cyanobacteria aqueous supernatant on cell line viability in vitro. However, the concentration that the test samples were administered was either not measured or not disclosed to the reader. Lines 168-169 suggest that this was natively diluted 1:100 in medium. The results are seemingly meaningless in that they cannot be compared to each other or anything else, because the concentration of natural products and other macromolecular components are unknown. Were the samples lyophilized to provide a dry weight that a wt/vol (e.g. μg/mL) concentration could be measured?

  Line 295 (Figure 3) has a distracting and confusing tree-like diagram for showing statistical significance of the difference of each sample supernatant compared to the control. Since these are all compared against the same column, it would be rather appropriate to put a *, **, or *** (or nothing), above every individual test sample from the cyanobacteria strains to clarify specifically which sample had what level of statistical significance compared to CTRL, with a brief definition of the symbols in the figure caption.

As a note, there is a landmark paper by Engene and colleagues about the phylogeny of filamentous cyanobacteria that would be very helpful for the authors to read for general knowledge and to reference in their manuscript. (Engene, N.; Gunasekera, S. P.; Gerwick, W. H.; Paul, V. J. Phylogenetic inferences reveal a large extent of novel biodiversity in chemically rich tropical marine cyanobacteria. Appl. Environ. Microbiol. 2013, 79, 1882–1888.)

One result and conclusion, specifically regarding strain ITAC105, is called into question for validity. In Figure 1 and Line 211, also described further on Lines 248-254, this organism is assigned as a Phormidium sp. with cells of diameter about 1 μm. I believe that this organism is more likely a Leptolyngbya sp. From the polyphasic approach, the morphology must be considered, and Phormidium are wider by about 3-15x. I can understand the desire to assign the genus by molecular methods, but it should also be considered that many Leptolyngbya organisms have been misclassified in the past, especially as Phormidium or even Lyngbya. For reference, one can read the book chapter by Komárek and colleagues from 2004, “Filamentous Cyanobacteria” that was published in Freshwater Algae of North America. The authors themselves of the current manuscript address this only vaguely and briefly on Lines 370-372 “… while previously ITAC105 was as[signed] to belong to the Leptolyngbya genus. It is further confusing that this organism is described in Table 1 as “undetermined Oscillatoriales” based on morphological features, but it really looks like Leptolyngbya to and was actually previously assigned according to Lines 370-372 and reference 28 by the same authorship as being a Leptolyngbya sp. If the authors are going to conclusively re-assign the taxonomy of this organism, it shall certainly merit more than one sentence saying so in the discussion section.

Lastly, the noted above “undetermined Oscillatoriales” should not be italicized because this is the name of an order and not a genus.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf