the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 08 Nov 2023
The calcitic test growth rate of Spirillina vivipara (Foraminifera)
Abstract. Foraminiferal groups encompass vastly differing morphologies, ecological preferences and elemental/ isotopic test compositions. For some of these groups, the calcification mechanism is completely unknown, but is likely differing from that of well-studied groups. This study aimed to understand the test growth and calcification rate of Spirillina vivipara Ehrenberg, 1843 (Order Spirillinida), and to compare them to other foraminifera species. Spirillinids have a closely coiled spiral chamber like a tube, yet their calcitic microstructure is unique amongst foraminifera. Calcification observations in S. vivipara facilitate the estimation of carbonate precipitation rates during active test elongation, which are revealed to be independent of the individual’s size. We found that S. vivipara grows its test in response to food availability, suggesting that calcification directly corresponds to cell growth. Timelapse observations of S. vivipara indicate continuous growth, suggesting active growth phases and interspersed rest periods, hinting at potential biological rhythms in the growth and calcification process. We also implemented a 24-hour observation period using Calcein staining, showing calcite precipitation rates of 8.08 µm/hour in S. vivipara, which correspond to approximately 364 nmol/cm2/min i.e. 36.4mg/cm2/min. These rates are higher than those published for other foraminifera and those in most inorganic precipitation experiments. Such high rates in spirillinid foraminifera may explain their distinctive morphology, elemental composition, unusual reproduction and ecological distribution.
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Notice on discussion status
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Preprint
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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(5905 KB) - Metadata XML
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Supplement
(288 KB) - BibTeX
- EndNote
- Final revised paper
Journal article(s) based on this preprint
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2023-2259', Anonymous Referee #1, 10 Dec 2023
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.
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AC1: 'Reply on RC1', Yukiko Nagai, 22 Jan 2024
Dear Reviewer #1,
We are grateful for the thorough review you have conducted on our manuscript and for the constructive feedback provided. We strongly agree with your remarks indicating that our application of microscopy techniques, such as FIB-SEM, along with the use of calcein staining, may contribute significantly to advancements in our understanding of foraminiferal calcification processes. The detailed comments included in the attached PDF are also highly appreciated. We shall revise the manuscript along with your comments.
In response to the issues raised, we plan to carefully revise the following modifications:
1) In the Introduction, we will expand on the ecology of Spirillina vivipara.
2) Within the Materials & Methods section, details regarding the calcein staining observations will be described.
3) We will specify the number of specimens used in our study. It is important to ensure transparency and reproducibility.
4) We acknowledge your valuable input on our statistical analyses. We will update the manuscript to include a revised description utilizing suggested Spearman’s correlation coefficient.
5) We will integrate descriptions of Figs. 3 and 4 into the relevant sections of the text to ensure that all figures are adequately discussed.
6) As for section 2.3, we will clarify the pretreatment procedures for FIB electron microscopy observations, correlating with the findings presented in Fig. 4. We believe these revisions will address your concerns and enhance the manuscript's clarity and precision.
Sincerely yours,
Yukiko Nagai
Citation: https://doi.org/10.5194/egusphere-2023-2259-AC1
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AC1: 'Reply on RC1', Yukiko Nagai, 22 Jan 2024
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RC2: 'Comment on egusphere-2023-2259', Delphine Dissard, 03 Jan 2024
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.
Citation: https://doi.org/10.5194/egusphere-2023-2259-RC2 -
AC2: 'Reply on Dr. Delphine Dissard', Yukiko Nagai, 22 Jan 2024
Dear Dr. Delphine Dissard,
We are profoundly grateful for the time and effort you have dedicated to peer-reviewing our manuscript. Your evaluation, recognizing our work as relevant within the scope of BG and addressing current scientific inquiries, is greatly encouraging.
As you have rightly noted, this paper has endeavored to estimate its calcification rate through a multitude of methodologies.
You have correctly pointed out that the text did not specify the number of samples used. Although we have included all relevant data within the tables, we acknowledge the necessity of clearly stating the sample sizes in the manuscript text. We will ensure this information is accurately incorporated.
Concerning the figures and tables, we should include the descriptions of Figs. 3 and 4 within the text. We would like to keep Tables 3 and 4 in the main manuscript, since they contain the original data of our measurements.
We also wish to express our gratitude for your attention to the time-lapse video of calcification. Your interest in this aspect of our work reinforces our belief in the video's contribution to the field.
The insights you have provided are invaluable, and they serve as a catalyst for enhancing the overall quality and clarity of our paper. We are committed to making the necessary amendments to ensure the manuscript is both accessible and enriched with information.
For minor comments, the answers are indicated below >.
Minor comments:
Line 158 refers to table 5 not 3
>Thanks for pointing this out, I will revise it to Table3.
Number of specimen used for calcification rate determination should definitely be included.
>We think you are correct. We will state the number of specimens.
Fig 3 and 4 not discussed in the text.
>Thank you for your suggestion. We will include description about Figures 3 and 4 to indicate the figures where appropriate in the text and discuss them.
line 31, references should be listed within the same bracket.
>Thank you for pointing out. We will correct the position of the bracket.
Warm regards,
Yukiko Nagai
Citation: https://doi.org/10.5194/egusphere-2023-2259-AC2
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AC2: 'Reply on Dr. Delphine Dissard', Yukiko Nagai, 22 Jan 2024
Interactive discussion
Status: closed
-
RC1: 'Comment on egusphere-2023-2259', Anonymous Referee #1, 10 Dec 2023
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.
-
AC1: 'Reply on RC1', Yukiko Nagai, 22 Jan 2024
Dear Reviewer #1,
We are grateful for the thorough review you have conducted on our manuscript and for the constructive feedback provided. We strongly agree with your remarks indicating that our application of microscopy techniques, such as FIB-SEM, along with the use of calcein staining, may contribute significantly to advancements in our understanding of foraminiferal calcification processes. The detailed comments included in the attached PDF are also highly appreciated. We shall revise the manuscript along with your comments.
In response to the issues raised, we plan to carefully revise the following modifications:
1) In the Introduction, we will expand on the ecology of Spirillina vivipara.
2) Within the Materials & Methods section, details regarding the calcein staining observations will be described.
3) We will specify the number of specimens used in our study. It is important to ensure transparency and reproducibility.
4) We acknowledge your valuable input on our statistical analyses. We will update the manuscript to include a revised description utilizing suggested Spearman’s correlation coefficient.
5) We will integrate descriptions of Figs. 3 and 4 into the relevant sections of the text to ensure that all figures are adequately discussed.
6) As for section 2.3, we will clarify the pretreatment procedures for FIB electron microscopy observations, correlating with the findings presented in Fig. 4. We believe these revisions will address your concerns and enhance the manuscript's clarity and precision.
Sincerely yours,
Yukiko Nagai
Citation: https://doi.org/10.5194/egusphere-2023-2259-AC1
-
AC1: 'Reply on RC1', Yukiko Nagai, 22 Jan 2024
-
RC2: 'Comment on egusphere-2023-2259', Delphine Dissard, 03 Jan 2024
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.
Citation: https://doi.org/10.5194/egusphere-2023-2259-RC2 -
AC2: 'Reply on Dr. Delphine Dissard', Yukiko Nagai, 22 Jan 2024
Dear Dr. Delphine Dissard,
We are profoundly grateful for the time and effort you have dedicated to peer-reviewing our manuscript. Your evaluation, recognizing our work as relevant within the scope of BG and addressing current scientific inquiries, is greatly encouraging.
As you have rightly noted, this paper has endeavored to estimate its calcification rate through a multitude of methodologies.
You have correctly pointed out that the text did not specify the number of samples used. Although we have included all relevant data within the tables, we acknowledge the necessity of clearly stating the sample sizes in the manuscript text. We will ensure this information is accurately incorporated.
Concerning the figures and tables, we should include the descriptions of Figs. 3 and 4 within the text. We would like to keep Tables 3 and 4 in the main manuscript, since they contain the original data of our measurements.
We also wish to express our gratitude for your attention to the time-lapse video of calcification. Your interest in this aspect of our work reinforces our belief in the video's contribution to the field.
The insights you have provided are invaluable, and they serve as a catalyst for enhancing the overall quality and clarity of our paper. We are committed to making the necessary amendments to ensure the manuscript is both accessible and enriched with information.
For minor comments, the answers are indicated below >.
Minor comments:
Line 158 refers to table 5 not 3
>Thanks for pointing this out, I will revise it to Table3.
Number of specimen used for calcification rate determination should definitely be included.
>We think you are correct. We will state the number of specimens.
Fig 3 and 4 not discussed in the text.
>Thank you for your suggestion. We will include description about Figures 3 and 4 to indicate the figures where appropriate in the text and discuss them.
line 31, references should be listed within the same bracket.
>Thank you for pointing out. We will correct the position of the bracket.
Warm regards,
Yukiko Nagai
Citation: https://doi.org/10.5194/egusphere-2023-2259-AC2
-
AC2: 'Reply on Dr. Delphine Dissard', Yukiko Nagai, 22 Jan 2024
Peer review completion
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Katsuyuki Uematsu
Briony Mamo
Takashi Toyofuku
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(5905 KB) - Metadata XML
-
Supplement
(288 KB) - BibTeX
- EndNote
- Final revised paper