Comments on the Ms “The calcitic test growth rate of Spirillina vivipara (Foraminifera)”

by Yukiko Nagai, Katsuyuki Uematsu, Briony Mamo, and Takashi Toyofuku

submitted for publication in Egusphere

Manuscript Number egusphere-2023-2259

General evaluation:

The subject of the paper falls in the general scope of Egusphere. The Ms focuses on understanding and evaluating the test growth rate of the foraminiferal species Spirillina vivipara under different culture conditions. The authors have provided compelling evidence on the calcification by using different techniques (e.g., microscopies like FIB-SEM) and methodologies (e.g., calcein) that should advance our knowledge on this domain. The Ms presents results of primary scientific research that have not previously published. The study is well executed but several inaccuracies are present, some sentences must be rephrased and parts rearranged in the text. As far as the minor comments I directly appended in the pdf version of the Ms itself, whereas specific  comments have been reported below:

1) the introduction would include the morphological description of S. vivipara as well as its biology and ecology;

2) M&M section lacks some important information such as calcein (green?);

3) the number of specimens and replicates would be included;

4) the statistical analysis would be likely redone. Pearson correlation is applied when data from both variables follow a normal distribution. Given the limited number of data (i.e., Figure 3), authors would have better used a Spearman’s rho correlation that has no normality (non-parametric test) assumptions. Please also clarify if reporting the coefficient of correlation or determination;

5) Figures 3 and 4 are not cited in the text;

6) paragraph 2.3 details the methodology for ultrastructure observations (i.e., TEM) that are not reported.

I hope these suggestions will assist the Editor in his/her decision and the authors to improve the Ms.

This study on the calcification rate of the high Magnesium benthic foraminifera Spirillina vivipara, falls within the scope of BG and addresses up to date scientific questions. This study reports on data acquired on living S. vivipara specimen first collected in the wild prior being maintained under culture experiments for different time period while submitted to precise observations (time-lapse images to measure test extension per unit of time) and staining protocols (calcein) in order to determine calcite deposition rate under different conditions (fed vs. non fed specimens). Through various state-of-the-art techniques this paper presents novel ideas and datas as a calcification rate for S. vivipara could be determined and was observed to be significantly higher than those of other foraminiferal species. The scientific methods used for S. vivipara calcification rate determination are clearly explained, and if the determination strategy can be subject of debate, the clear description of the used technique should allow comparison with future calcification rates to be determined on other foraminifera species, which always remain a challenge. In parallel and a fortiori, such description should allow the reproduction of the described observations by fellow scientists.

A major downfall though is the lack of description on the number of specimens involved and replicates considered which needs to be added clearly within this manuscript. I am also not convinced about the results of statistical analyses on such a small dataset, hence the precise description of the data set should help the reader to understand the depth of described statistical results. Number and significance of figures are ok, but maybe table 3 and 4 would rather belong to the supplementary material. Similarly Fig. 3 and 4 are not discussed within the manuscript and should therefore either be removed or described. Finally, the two videos of time-lapse images that can be found in the supplementary material are welcome and of great interest. To conclude I recommend the publication of this manuscript after minor revisions.

Minor comments: 

Ligne 158 refers to table 5 not 3

Number of specimen used for calcification rate determination should definitely be included.

Fig 3 and 4 not discussed in the text.

line 31, references should be listed within the same bracket.