the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 05 May 2021
Fluorescent double labelling of F-actin in Foraminifera: evaluation of granular pattern F-actin organisation in reticulopodia
Abstract. This paper presents novel results of experiments focused on actin cytoskeleton organisation in pseudopodial structures of foraminifera. The main aim of this research was to test the hypotheses proposed to explain the previously reported granular pattern of F-actin labelling in several species of foraminifera with SiR-actin fluorescent probe. These hypotheses include the possibility that SiR-actin enhances the F-actin polymerisation or that it binds to other than F-actin compounds within foraminiferal cells, resulting in capturing the staining artefacts. The series of replicated experiments conducted on small miliolid Quinqueloculina sp. included double staining of F-actin with SiR-actin and phalloidin-based probes, complemented with observations of reticulopodia under the polarised light to identify the granules showing birefringence. All of performed experiments demonstrate highly congruent results expressed in SiR-actin and phalloidin co-labelling patterns, especially pronounced with the small granular objects (SiR-actin labelled granules or ALGs). Furthermore, the birefringence strongly tends to characterise primarily areas stained with both of the probes. These results rule out staining artefacts and support existence of actin cytoskeleton associated with micrometre-size motile granules. We discuss possible implications of this granular pattern that facilitates efficient formation and fast reorganization of extended granuloreticulopodia and other pseudopodial structures. If this is the case, they are one of the key evolutionary adaptations of these organisms that most likely predates the evolution of the foraminiferal tests that started in the early Palaeozoic.
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RC1: 'Comment on bg-2021-76', Jutta Wollenburg, 20 May 2021
The manuscript by Golén et al. is the logical consequence of their previous paper (Golén et al., 2020) where they observed a very distinct F-actin labelling in foraminiferal granule. In this new paper they applied co-labelling with SiR-actin and phalloidin on pseudopodial structure to rule out SiR-actin staining artefacts as a possible cause for the intense F-actin labellin described be Golén et al (2020). The paper shows impressive and stunning results and I have no doubts in the methods. However, the paper is unprecise in how many species and how many specimens were finally successfully investigated with this double staining method. If I am not wrong, only one Quinqueloculina specimen and only one area of its pseudopodial network is shown in the figures investigated. These things need to be clearified. I assume it was a tremendous effort and most attempts failed, anyhow, these are things that are important to show. I suggest to create a table with all species and the number of specimens involved, showing then which steps were successful, which failed, and how many specimens of which species could/was finally investigated for its fluorescence. Having eventually just one positive outcome is no problem but the reader want’s to know.
I sometimes had problems to understand the meaning of sentences, thus, I think the revised version should be read by a native speaker before submission.
The Pdf file lacks line numbers which complicates the review
Some further comments
Introduction
The first three sentences lack any references and give incomplete statements. Please streamline these aspects, provide information on the differences in pseudopodia, what is known about the content of granule etc. , and add references on these topics. I would also recommend that the first sentence is written in a way to provide the none-foram reader with some basic informations on what pseudopodia are and for which purposes they are used. Provide some background on granule, historical and information on the kind of granule that exist according to literature. Either here or when you use them, you have to explain terms like ‘trunk reticulopodia’ as, I guess the majority of readers may not be familiar with such terms.
2 Material and methods
2.1 Specimens
It is irrelevant how many species are in this tank, it just matters how many were used/investigated for this study.
It would be interesting to know which Quinqueloculina species was used and which additional species, not just the genus, were investigated.
The statement ‘This taxon presented the most stable and predictable reticulopodial activity , thus, was most suitable for replicated experiments.’ I would suggest this is a result. I would also modify it in the ‘bold’ way. I am not sure what you wanted to say with the last sentence of this paragraph.
2.3 Fixation
You describe that you optimised the fixation method by the trial and error method. It would be good to provide all respective receipts and the results following their application. The reader may want to apply the method in her/his own research and don’t have face the same failures. Please also provide the specifications for the glutaraldehyde and what else is used (e.g. Cacodylbuffer??, millepore filtered water??,…). Describe how the fixative was washed out etc.
Later in the text you write ‘It is likely that standard fixation methods make ALGs very difficult to preserve during fixation.’ It would be good for the discussion to show the fixation receipts in previous and this manuscript/-s, perhaps in the supplements.
2.4 Staining
About the same issue as before. Please provide details. I guess you mixed a stock solution, then stored it at xy temperature, then you applied the staining by adding xy µl of the stock solution to xy ml in your petri dish… How did you wash out the dye? How did you dry the samples? Etc.
2.5 Imaging
‘If necessary, fluorescence images were processed using FIJI software to remove the background noise.’ Please explain what this software does.
‘Additionally, imaging of the pseudopodium of an unstained individual with the same light source intensity 130 and exposure time was done to control autofluorescence.’ Was this species and its pseudopods fixed in the same way?
- Results
Please rewrite the subchapter 3.3 (should be 3.1) Control for autofluorescence
‘Profile of the intensity of fluorescence along the line that crosses the pseudopodium shows low level of the fluorescence intensity in the unstained (control) individual.’ Please rewrite starting with ‘In the unstained control foraminifera (provide species) the fluorescence intensity profile ..´. I have no idea what you wanted to say. If possible provide a figure or refer to a respective figure for the control specimen (just one???). ´ Also in this individual the variability of the intensity is low, there is no significant difference between the pseudopodium and the background´ Observed with which channels?
´The individual labelled with SiR-actin and Phalloidin Atto 488 displays much higher intensity levels and variation of the intensity with the intensity peaks in the same location. Please be precise, which channels were used for the control, which channels for which dye in which specimen of which species. Refer to the respective figures.
`The relative height of the peak is larger for the SiR actin channel (Fig. S3 in Supplementary Materials).´…is larger for the SiR … than for…
3.2 Colocalisation of signal from SiR-actin and Phalloidin Atto 488 in fluorescently stained pseudopodia
I really have big problems in understanding some of the sentences in this section. It would be good if a native speaker reads and eventually corrects some parts of the revised manuscript. ‘Moreover, z-position in which given object appeared the sharpest in the Phalloidin Atto was shifted away from the objective for about 620-930 nm in relation to the z-position in which the same object was in focus in the SiR-actin channel .’ I have no idea what is written here, please rewrite. ‘The light of different wavelength is focused at different positions as refractive index of a medium depends on the wave length (Stanley 1971).’ Please rewrite. ‘Both probes stained the most intensely the granular objects (see Fig. 1), however, the whole reticulopodium also displayed weaker fluorescence.’ Are you talking about autofluorescence or is this fluorescence related to the stains?
‘The relative intensity of signal in this area compared to the intensity of fluorescence throughout the whole pseudopodium appeared to be higher for Phalloidin Atto 488 than for SiR-actin.’ Do you mean ‘In this area fluorescence intensity for Phalloidin Atto 488 is much higher than for SiR-actin, whereas, in the remaining pseudopodium…?’
Some exemplified typos
Introduction
.. which elaborate hierarchical
..critical in understanding the evolution
.. see (Goldstein, 1999).
.. fluorescence microscopy
..fixed specimens of various species
the classical method of imaging
.. the classical method for imaging
… In an attempt to verify if the possibility that these filaments are the form of are actin they tried to label them with myosin sub-fragment 1 (myosin S1). As these filaments in question did not bind myosin S1, they concluded that the nature of these structures differ from the actin.
.. Koonce et al. (1986b) demonstrated by fluorescent imaging, using the rhodamine-phalloidin, the presence of microfilaments spread throughout all pseudopods.
Results starts with number 3.3.
Fig. legends, number 2 is missing but there is two times fig. 1.
Citation: https://doi.org/10.5194/bg-2021-76-RC1 -
AC1: 'Reply on RC1', Jan Goleń, 15 Jun 2021
Response to the interactive comment by Jutta Wollenburg.
On behalf of all authors I we would like to thank Dr Jutta Wollenburg for her constructive comments. We are preparing a detailed response to all the comments to be submitted as soon as possible after the end of open discussion. At the moment I would like to refer to two most important and general issues raised by the referee.
The first issue is the number of the individuals presented in the paper. The images presented in the submitted manuscript are based only on two individuals. However, all replicated experiments have been done on much higher number of individuals. We agree that it is important to give a precise number of individuals presented in the paper and we will clarify it in the revised version of the manuscript. We chose those two individuals because the preservation of the granuloreticulopodial structures was the best. and We will do our best to provide as good as possible documentation with new figures added to the main text and/or to supplementary materials.
The second issue is the taxonomic attribution of the presented individuals. To identify the species or genera of individuals used in the study we consulted the published data on the species composition in the sea-water aquarium in Burgers’ Zoo (Arnhem, the Netherlands) (Ernst et al., 2011). This paper presents SEM images of the individuals identified samples from this aquarium, individual no. 5 in fig. 4 most closely resembles the specimens used in or study. This individual was described by Ernst et al. (2011) as belonging to the genus Quinqueloculina, it lacks identification at the species level. Overall we observe similar diversity of miliolids in our sample as in Ernst et al. (2011). Following your comment and suggestion, we would like to admit that taxonomy is, in general, a truly important matter, therefore, we are working on extra documentation of the studied individuals under the SEM. We hope that such documentation should allow for accurate identification of the species.
All other comments will be addressed in our detailed response. We would like to submit the detailed response at the beginning of July within the 3 weeks’ period from the end of the open discussion.
We are very grateful for the helpful and insightful comments.
Kind regards,
Jan Goleń
Citation: https://doi.org/10.5194/bg-2021-76-AC1 - AC3: 'Reply on RC1', Jan Goleń, 07 Jul 2021
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RC2: 'Comment on bg-2021-76', Takashi Toyofuku, 30 May 2021
General comment
This research investigated a point that was unclear in the previously published research. Although the content is good, it is questionable whether the purpose and discussion match the common interests of the audience of this journal. In addition, I think many points can be discussed with previous studies, but they are lacking. If these points are improved, I can agree there is a great possibility that the contents of this paper can be published in Biogeosciences.
L 21 Introduction
"…To account for this possibility, the term SiR-actin labelled granules has been coined to describe them (GolenÌ et al. 2020). The presented study primarily addresses the question, whether they are experimental artefacts or they represent physiological and functional forms of F-actin in foraminifera."
This targeting is too specific and does not contribute to the general readership.
Too much value is placed on the biological perspective for this study. It will be classified as a more biological and protozoan work. The discussion should return to the proposition that "Foraminifera pseudopodia are very important for understanding the evolution, morphogenesis, physiology, and ecology of these organisms." Otherwise, the position of this research in Biogeoscience and Earthscience will be unclear. It is essential to discuss this research and insights into biomineralization and shell morphology. Considering that cytoskeletal variation governs pseudopod extension and that the three-dimensional structure of the pseudopod unfolding from the aperture governs shell morphology (Tyszka et al, 2005; Tyszka, 2006), it should not be too difficult to connect and discuss the results of this study with these perspectives.
Method
L90.
If possible, can the origin of the samples be shown? I would imagine, however, that foraminifera would have been introduced mixed in with corals and other macro-organisms from various origins. Since authors can not know where they originated, it seems like a good idea to identify the species or provide SEM photos. I do not think there is any need to hesitate on account of the deformity. From my own research experience, I am aware that the shell morphology of Miliolid is easily affected in captive environments.
L.128
Add "Digital Single Lens Reflex camera" around Cannon DS 126231
L. 133
3.3 Control for autofluorescence →3.1 Control for autofluorescence
L. 140
"The staining of reticulopodium with both of fluorescent probes was successful "
Describe and discuss the reasons and conditions for what authors would call a "successful." Just quoting Figure 1-3 is not enough explanation, and the reader will not know if it is successful or not.
L. 334
Figure 1→Figure 2
L. 145
"Stanley 1971"
This study focuses on the localization identity, and it is a fundamental issue of this study, then needs a solid evaluation, rather than discussing the possibility ("possibly" in L144).
If this can be technically corrected, and it can be proved that there is no problem, then it is better to state in the method "analyzed by correcting for differences in depth of focus depending on wavelength" and describe the correction methodology in the supplement.
This study focuses on the localization identity, and it is a fundamental issue of this study.
L. 159
3.3 MT
Abbreviations that appear for the first time should be accompanied by an explanation.
L161
"The fact that three independent methods indicate presence of the F-actin …"
What are the three methods, SiR-Actin, Phalloidin Atto 488, and birefringence? I believe that the authors have shown that they existed in the same place. However, it is a leap to say that this is the basis for showing that F-actin is present in the reticulopodium. Authors need to explain it sequentially to complete the logic for example.
The signal of SiR-Acrin was detected orthotopically with that of Phalloidin Atto 488. Phalloidin is known to bind specifically to F-actin and is used as a major indicator substrate for F-actin. (Cooper, 1987 DOI: 10.1083/jcb.105.4.1473 ). It is also known that F-actin bundles exhibit birefringence. (Hodge AJ. J Biophys Biochem Cytol. 1955 Jul 25;1(4):361-80. DOI: 10.1083/JCB.1.4.361; Hodge AJ. J Biophys Biochem Cytol. 1956 Jul 25;2(4 Suppl):131-42. DOI: 10.1083/jcb.2.4.131. These results strongly suggest that the signal of SiR-Actin indicates F-actin.
The above is an example, but this is the kind of discussion that needs to be addressed. This is the primary purpose of this paper. Further, TEM images of granular materials are also demanded in future studies. After that, we can start to discuss whether granular materials have much F-actin or not.
L. 195 "suggest that they are key evolutionary adaptation that most likely predated emergence of foraminiferal tests in the early Palaeozoic."
The results of Pawloski et al. (2013: already referred to in this study) should be cited and discussed. It is also essential to compare the results with Habura et al. (2005: https://doi.org/10.1093/molbev/msi190), who attempted to explain the quick movements of pseudopodia from the aspect of Tubulin.
L. 196 "They probably facilitate efficient formation of tests and fast reorganization of pseudopodial structures in Foraminifera. "
Provide evidence for why authors think so, and discuss the connection to shell formation.
L. 198 cannot be determined without more detailed ultrastructural studies
I agree with this statement, but there are many examples of previous studies that have observed the movement, function, and microstructure of pseudopods during shell formation. Based on the present findings, a discussion of the contents of these previous studies must be made. In particular, I believe that comparisons with and interpretations of the authors' previous studies can be made with certainty.
Citation: https://doi.org/10.5194/bg-2021-76-RC2 -
AC2: 'Reply on RC2', Jan Goleń, 15 Jun 2021
Response to the interactive comment by Takashi Toyofuku.
On behalf of all authors I we would like to thank Dr Takashi Toyofuku for his constructive comments. We are preparing a detailed response to all the comments that should be submitted as soon as possible after the end of open discussion. At the moment I would like to address the most crucial issues which are (1) the suitability of the submitted paper to the journal, (2) the proper origin of the samples, the species identification and the SEM documentation.
The journal Biogeosciences has published numerous papers concerning foraminifera, including many studies of the physiological processes involved in biomineralisation and morphogenesis of the foraminiferal tests. Actin cytoskeleton is highly involved in various aspects of the chamber formation (Tyszka et al. 2019), hence the issue of organisation of actin has implications for understanding of these processes. Our study is within the scope of the journal, but we agree that the logical connection to the geosciences should be emphasised more clearly. We are very grateful for raising this question and suggestion of the literature concerning the in silico modelling of the morphogenesis of shells. In the revised version of the manuscript we plan to add suggested information followed by relevant references, including the ones pointed out by the referee.
Concerning the second point, it is true that samples organisms within the marine aquaria in the Burgers’ Zoo originate from various locations, so the geographic area of origin cannot be narrowed down more precisely than to the Indo-Pacific region. We agree with the referee’s suggestion that for this reason additional SEM documentation would be of great value, allowing for the more accurate species identification. We are performing additional documentation, including SEM imaging.
All other comments are also greatly appreciated and will be addressed in a detailed response. We would like to submit the detailed response at the beginning of July within the 3 weeks’ period from the end of the open discussion stage.
Kind regards,Jan Goleń
Citation: https://doi.org/10.5194/bg-2021-76-AC2 - AC4: 'Reply on RC2', Jan Goleń, 07 Jul 2021
-
AC2: 'Reply on RC2', Jan Goleń, 15 Jun 2021
Status: closed
-
RC1: 'Comment on bg-2021-76', Jutta Wollenburg, 20 May 2021
The manuscript by Golén et al. is the logical consequence of their previous paper (Golén et al., 2020) where they observed a very distinct F-actin labelling in foraminiferal granule. In this new paper they applied co-labelling with SiR-actin and phalloidin on pseudopodial structure to rule out SiR-actin staining artefacts as a possible cause for the intense F-actin labellin described be Golén et al (2020). The paper shows impressive and stunning results and I have no doubts in the methods. However, the paper is unprecise in how many species and how many specimens were finally successfully investigated with this double staining method. If I am not wrong, only one Quinqueloculina specimen and only one area of its pseudopodial network is shown in the figures investigated. These things need to be clearified. I assume it was a tremendous effort and most attempts failed, anyhow, these are things that are important to show. I suggest to create a table with all species and the number of specimens involved, showing then which steps were successful, which failed, and how many specimens of which species could/was finally investigated for its fluorescence. Having eventually just one positive outcome is no problem but the reader want’s to know.
I sometimes had problems to understand the meaning of sentences, thus, I think the revised version should be read by a native speaker before submission.
The Pdf file lacks line numbers which complicates the review
Some further comments
Introduction
The first three sentences lack any references and give incomplete statements. Please streamline these aspects, provide information on the differences in pseudopodia, what is known about the content of granule etc. , and add references on these topics. I would also recommend that the first sentence is written in a way to provide the none-foram reader with some basic informations on what pseudopodia are and for which purposes they are used. Provide some background on granule, historical and information on the kind of granule that exist according to literature. Either here or when you use them, you have to explain terms like ‘trunk reticulopodia’ as, I guess the majority of readers may not be familiar with such terms.
2 Material and methods
2.1 Specimens
It is irrelevant how many species are in this tank, it just matters how many were used/investigated for this study.
It would be interesting to know which Quinqueloculina species was used and which additional species, not just the genus, were investigated.
The statement ‘This taxon presented the most stable and predictable reticulopodial activity , thus, was most suitable for replicated experiments.’ I would suggest this is a result. I would also modify it in the ‘bold’ way. I am not sure what you wanted to say with the last sentence of this paragraph.
2.3 Fixation
You describe that you optimised the fixation method by the trial and error method. It would be good to provide all respective receipts and the results following their application. The reader may want to apply the method in her/his own research and don’t have face the same failures. Please also provide the specifications for the glutaraldehyde and what else is used (e.g. Cacodylbuffer??, millepore filtered water??,…). Describe how the fixative was washed out etc.
Later in the text you write ‘It is likely that standard fixation methods make ALGs very difficult to preserve during fixation.’ It would be good for the discussion to show the fixation receipts in previous and this manuscript/-s, perhaps in the supplements.
2.4 Staining
About the same issue as before. Please provide details. I guess you mixed a stock solution, then stored it at xy temperature, then you applied the staining by adding xy µl of the stock solution to xy ml in your petri dish… How did you wash out the dye? How did you dry the samples? Etc.
2.5 Imaging
‘If necessary, fluorescence images were processed using FIJI software to remove the background noise.’ Please explain what this software does.
‘Additionally, imaging of the pseudopodium of an unstained individual with the same light source intensity 130 and exposure time was done to control autofluorescence.’ Was this species and its pseudopods fixed in the same way?
- Results
Please rewrite the subchapter 3.3 (should be 3.1) Control for autofluorescence
‘Profile of the intensity of fluorescence along the line that crosses the pseudopodium shows low level of the fluorescence intensity in the unstained (control) individual.’ Please rewrite starting with ‘In the unstained control foraminifera (provide species) the fluorescence intensity profile ..´. I have no idea what you wanted to say. If possible provide a figure or refer to a respective figure for the control specimen (just one???). ´ Also in this individual the variability of the intensity is low, there is no significant difference between the pseudopodium and the background´ Observed with which channels?
´The individual labelled with SiR-actin and Phalloidin Atto 488 displays much higher intensity levels and variation of the intensity with the intensity peaks in the same location. Please be precise, which channels were used for the control, which channels for which dye in which specimen of which species. Refer to the respective figures.
`The relative height of the peak is larger for the SiR actin channel (Fig. S3 in Supplementary Materials).´…is larger for the SiR … than for…
3.2 Colocalisation of signal from SiR-actin and Phalloidin Atto 488 in fluorescently stained pseudopodia
I really have big problems in understanding some of the sentences in this section. It would be good if a native speaker reads and eventually corrects some parts of the revised manuscript. ‘Moreover, z-position in which given object appeared the sharpest in the Phalloidin Atto was shifted away from the objective for about 620-930 nm in relation to the z-position in which the same object was in focus in the SiR-actin channel .’ I have no idea what is written here, please rewrite. ‘The light of different wavelength is focused at different positions as refractive index of a medium depends on the wave length (Stanley 1971).’ Please rewrite. ‘Both probes stained the most intensely the granular objects (see Fig. 1), however, the whole reticulopodium also displayed weaker fluorescence.’ Are you talking about autofluorescence or is this fluorescence related to the stains?
‘The relative intensity of signal in this area compared to the intensity of fluorescence throughout the whole pseudopodium appeared to be higher for Phalloidin Atto 488 than for SiR-actin.’ Do you mean ‘In this area fluorescence intensity for Phalloidin Atto 488 is much higher than for SiR-actin, whereas, in the remaining pseudopodium…?’
Some exemplified typos
Introduction
.. which elaborate hierarchical
..critical in understanding the evolution
.. see (Goldstein, 1999).
.. fluorescence microscopy
..fixed specimens of various species
the classical method of imaging
.. the classical method for imaging
… In an attempt to verify if the possibility that these filaments are the form of are actin they tried to label them with myosin sub-fragment 1 (myosin S1). As these filaments in question did not bind myosin S1, they concluded that the nature of these structures differ from the actin.
.. Koonce et al. (1986b) demonstrated by fluorescent imaging, using the rhodamine-phalloidin, the presence of microfilaments spread throughout all pseudopods.
Results starts with number 3.3.
Fig. legends, number 2 is missing but there is two times fig. 1.
Citation: https://doi.org/10.5194/bg-2021-76-RC1 -
AC1: 'Reply on RC1', Jan Goleń, 15 Jun 2021
Response to the interactive comment by Jutta Wollenburg.
On behalf of all authors I we would like to thank Dr Jutta Wollenburg for her constructive comments. We are preparing a detailed response to all the comments to be submitted as soon as possible after the end of open discussion. At the moment I would like to refer to two most important and general issues raised by the referee.
The first issue is the number of the individuals presented in the paper. The images presented in the submitted manuscript are based only on two individuals. However, all replicated experiments have been done on much higher number of individuals. We agree that it is important to give a precise number of individuals presented in the paper and we will clarify it in the revised version of the manuscript. We chose those two individuals because the preservation of the granuloreticulopodial structures was the best. and We will do our best to provide as good as possible documentation with new figures added to the main text and/or to supplementary materials.
The second issue is the taxonomic attribution of the presented individuals. To identify the species or genera of individuals used in the study we consulted the published data on the species composition in the sea-water aquarium in Burgers’ Zoo (Arnhem, the Netherlands) (Ernst et al., 2011). This paper presents SEM images of the individuals identified samples from this aquarium, individual no. 5 in fig. 4 most closely resembles the specimens used in or study. This individual was described by Ernst et al. (2011) as belonging to the genus Quinqueloculina, it lacks identification at the species level. Overall we observe similar diversity of miliolids in our sample as in Ernst et al. (2011). Following your comment and suggestion, we would like to admit that taxonomy is, in general, a truly important matter, therefore, we are working on extra documentation of the studied individuals under the SEM. We hope that such documentation should allow for accurate identification of the species.
All other comments will be addressed in our detailed response. We would like to submit the detailed response at the beginning of July within the 3 weeks’ period from the end of the open discussion.
We are very grateful for the helpful and insightful comments.
Kind regards,
Jan Goleń
Citation: https://doi.org/10.5194/bg-2021-76-AC1 - AC3: 'Reply on RC1', Jan Goleń, 07 Jul 2021
-
RC2: 'Comment on bg-2021-76', Takashi Toyofuku, 30 May 2021
General comment
This research investigated a point that was unclear in the previously published research. Although the content is good, it is questionable whether the purpose and discussion match the common interests of the audience of this journal. In addition, I think many points can be discussed with previous studies, but they are lacking. If these points are improved, I can agree there is a great possibility that the contents of this paper can be published in Biogeosciences.
L 21 Introduction
"…To account for this possibility, the term SiR-actin labelled granules has been coined to describe them (GolenÌ et al. 2020). The presented study primarily addresses the question, whether they are experimental artefacts or they represent physiological and functional forms of F-actin in foraminifera."
This targeting is too specific and does not contribute to the general readership.
Too much value is placed on the biological perspective for this study. It will be classified as a more biological and protozoan work. The discussion should return to the proposition that "Foraminifera pseudopodia are very important for understanding the evolution, morphogenesis, physiology, and ecology of these organisms." Otherwise, the position of this research in Biogeoscience and Earthscience will be unclear. It is essential to discuss this research and insights into biomineralization and shell morphology. Considering that cytoskeletal variation governs pseudopod extension and that the three-dimensional structure of the pseudopod unfolding from the aperture governs shell morphology (Tyszka et al, 2005; Tyszka, 2006), it should not be too difficult to connect and discuss the results of this study with these perspectives.
Method
L90.
If possible, can the origin of the samples be shown? I would imagine, however, that foraminifera would have been introduced mixed in with corals and other macro-organisms from various origins. Since authors can not know where they originated, it seems like a good idea to identify the species or provide SEM photos. I do not think there is any need to hesitate on account of the deformity. From my own research experience, I am aware that the shell morphology of Miliolid is easily affected in captive environments.
L.128
Add "Digital Single Lens Reflex camera" around Cannon DS 126231
L. 133
3.3 Control for autofluorescence →3.1 Control for autofluorescence
L. 140
"The staining of reticulopodium with both of fluorescent probes was successful "
Describe and discuss the reasons and conditions for what authors would call a "successful." Just quoting Figure 1-3 is not enough explanation, and the reader will not know if it is successful or not.
L. 334
Figure 1→Figure 2
L. 145
"Stanley 1971"
This study focuses on the localization identity, and it is a fundamental issue of this study, then needs a solid evaluation, rather than discussing the possibility ("possibly" in L144).
If this can be technically corrected, and it can be proved that there is no problem, then it is better to state in the method "analyzed by correcting for differences in depth of focus depending on wavelength" and describe the correction methodology in the supplement.
This study focuses on the localization identity, and it is a fundamental issue of this study.
L. 159
3.3 MT
Abbreviations that appear for the first time should be accompanied by an explanation.
L161
"The fact that three independent methods indicate presence of the F-actin …"
What are the three methods, SiR-Actin, Phalloidin Atto 488, and birefringence? I believe that the authors have shown that they existed in the same place. However, it is a leap to say that this is the basis for showing that F-actin is present in the reticulopodium. Authors need to explain it sequentially to complete the logic for example.
The signal of SiR-Acrin was detected orthotopically with that of Phalloidin Atto 488. Phalloidin is known to bind specifically to F-actin and is used as a major indicator substrate for F-actin. (Cooper, 1987 DOI: 10.1083/jcb.105.4.1473 ). It is also known that F-actin bundles exhibit birefringence. (Hodge AJ. J Biophys Biochem Cytol. 1955 Jul 25;1(4):361-80. DOI: 10.1083/JCB.1.4.361; Hodge AJ. J Biophys Biochem Cytol. 1956 Jul 25;2(4 Suppl):131-42. DOI: 10.1083/jcb.2.4.131. These results strongly suggest that the signal of SiR-Actin indicates F-actin.
The above is an example, but this is the kind of discussion that needs to be addressed. This is the primary purpose of this paper. Further, TEM images of granular materials are also demanded in future studies. After that, we can start to discuss whether granular materials have much F-actin or not.
L. 195 "suggest that they are key evolutionary adaptation that most likely predated emergence of foraminiferal tests in the early Palaeozoic."
The results of Pawloski et al. (2013: already referred to in this study) should be cited and discussed. It is also essential to compare the results with Habura et al. (2005: https://doi.org/10.1093/molbev/msi190), who attempted to explain the quick movements of pseudopodia from the aspect of Tubulin.
L. 196 "They probably facilitate efficient formation of tests and fast reorganization of pseudopodial structures in Foraminifera. "
Provide evidence for why authors think so, and discuss the connection to shell formation.
L. 198 cannot be determined without more detailed ultrastructural studies
I agree with this statement, but there are many examples of previous studies that have observed the movement, function, and microstructure of pseudopods during shell formation. Based on the present findings, a discussion of the contents of these previous studies must be made. In particular, I believe that comparisons with and interpretations of the authors' previous studies can be made with certainty.
Citation: https://doi.org/10.5194/bg-2021-76-RC2 -
AC2: 'Reply on RC2', Jan Goleń, 15 Jun 2021
Response to the interactive comment by Takashi Toyofuku.
On behalf of all authors I we would like to thank Dr Takashi Toyofuku for his constructive comments. We are preparing a detailed response to all the comments that should be submitted as soon as possible after the end of open discussion. At the moment I would like to address the most crucial issues which are (1) the suitability of the submitted paper to the journal, (2) the proper origin of the samples, the species identification and the SEM documentation.
The journal Biogeosciences has published numerous papers concerning foraminifera, including many studies of the physiological processes involved in biomineralisation and morphogenesis of the foraminiferal tests. Actin cytoskeleton is highly involved in various aspects of the chamber formation (Tyszka et al. 2019), hence the issue of organisation of actin has implications for understanding of these processes. Our study is within the scope of the journal, but we agree that the logical connection to the geosciences should be emphasised more clearly. We are very grateful for raising this question and suggestion of the literature concerning the in silico modelling of the morphogenesis of shells. In the revised version of the manuscript we plan to add suggested information followed by relevant references, including the ones pointed out by the referee.
Concerning the second point, it is true that samples organisms within the marine aquaria in the Burgers’ Zoo originate from various locations, so the geographic area of origin cannot be narrowed down more precisely than to the Indo-Pacific region. We agree with the referee’s suggestion that for this reason additional SEM documentation would be of great value, allowing for the more accurate species identification. We are performing additional documentation, including SEM imaging.
All other comments are also greatly appreciated and will be addressed in a detailed response. We would like to submit the detailed response at the beginning of July within the 3 weeks’ period from the end of the open discussion stage.
Kind regards,Jan Goleń
Citation: https://doi.org/10.5194/bg-2021-76-AC2 - AC4: 'Reply on RC2', Jan Goleń, 07 Jul 2021
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AC2: 'Reply on RC2', Jan Goleń, 15 Jun 2021
Video supplement
Movie S2. Z-stack of granuloreticulopodia of Quinqueloculina sp. Goleń, Jan; Tyszka, Jarosław; Godos, Karolina https://av.tib.eu/media/51971
Movie S1. Fixation of granuloreticulopodia of Amphistegina lessonii d’Orbigny Goleń, Jan; Tyszka, Jarosław; Godos, Karolina https://doi.org/10.5446/51970
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