Author Response
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Reviewer #1 (Public Review):
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Summary:
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The manuscript by Dubicka and co-workers on calcification in miliolid foraminifera presents an interesting piece of work. The study uses confocal and electron microscopy to show that the traditional picture of calcification in porcelaneous foraminifera is incorrect.
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Strengths:
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The authors present high-quality images and an original approach to a relatively solid (so I thought) model of calcification.
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Weaknesses:
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There are several major shortcomings. Despite the interesting subject and the wonderful images, the conclusions of this manuscript are simply not supported at all by the results. The fluorescent images may not have any relation to the process of calcification and should therefore not be part of this manuscript. The SEM images, however, do point to an outdated idea of miliolid calcification. I think the manuscript would be much stronger with the focus on the SEM images and with the speculation of the physiological processes greatly reduced.
</blockquote>
Reply: We would like to give thanks for all of the highly valuable comments. Prior to our study, we were also convinced that the calcification model of Miliolid (porcelaneous) foraminifera was relatively solid. Nevertheless, our SEM imaging results surprisingly contradicted the old model. The main difference is the in situ biomineralization of calcitic needles that precipitate within the chamber wall after deposition of ACC-bearing vesicles. We agree that our fluorescence studies presented in the paper are not conclusive evidence for the calcification model used by the studied Miliolid species. However, our fluorescent results show that “the old model” (sensu Hemleben et al., 1986) is not completely outdated. Most of the fluorescent imaging data show a vesicular transport of substrates necessary for calcification. This transport is presented by Calcein labelling experiments (Movie 1 that show a high number of dynamic endocytic vesicles of sea water circulation within the cytoplasm. These very fine Calcein-labelled vesicles are most likely responsible for transport and deposition of Ca2+ ions. This is partly consistent with the model presented by Hemleben et al. (1986). We may speculate that calcite nucleation is already occurring within the transported vesicles, but at this stage of research we have no evidence for this phenomenon.
Further live imaging fluorescence data show autofluorescence of vesicles upon excitation at 405 nm (emission 420–480 nm) associated with acidic vesicles marked by pH-sensitive LysoGlow84, may be a hint indicating association of ACC-bearing vesicles with acidic vesicles. Such spatial association of these vesicles may indicate a mechanism of pH elevation in the vesicles transporting Ca2+-rich gel to the calcifying wall of the new chamber.
We will do our best to limit the physiological interpretation presented based on fluorescence studies in the revised version of the manuscript. We are convinced that our fluorescent live imaging experiments provide important observations in biomineralizing Miliolid foraminifera, which are still missing in the existing literature. It should be stressed that all the fluorescent experiments and SEM observations were based on specimens constructing and biomineralizing new chambers. All of them belong to the same species and come from the same culture. Due to the aforementioned reasons, it is worthwhile presenting these complimentary results of our study. In the future they may be helpful in further exploration and understanding of all aspects of calcification in foraminifera.
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Reviewer #2 (Public Review):
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Summary:
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Dubicka et al. in their paper entitled " Biocalcification in porcelaneous foraminifera" suggest that in contrast to the traditionally claimed two different modes of test calcification by rotallid and porcelaneous miliolid formaminifera, both groups produce calcareous tests via the intravesicular mineral precursors (Mg-rich amorphous calcium carbonate). These precursors are proposed to be supplied by endocytosed seawater and deposited in situ as mesocrystals formed at the site of new wall formation within the organic matrix. The authors did not observe the calcification of the needles within the transported vesicles, which challenges the previous model of miliolid mineralization. Although the authors argue that these two groups of foraminifera utilize the same calcification mechanism, they also suggest that these calcification pathways evolved independently in the Paleozoic.
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Reply: We would like to acknowledge the review and all valuable comments. We do not argue that Miliolida and Rotallida utilise an identical calcification mechanism, but both groups utilize less divergent crystallization pathways, where mesocrystalline chamber walls are created by accumulating and assembling particles of pre-formed liquid amorphous mineral phase.
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Strengths:
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The authors document various unknown aspects of calcification of Pseudolachlanella eburnea and elucidate some poorly explained phenomena (e.g., translucent properties of the freshly formed test) however there are several problematic observations/interpretations which in my opinion should be carefully addressed.
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Weaknesses:
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<ol>
<li>The authors (line 122) suggest that "characteristic autofluorescence indicates the carbonate content of the vesicles (Fig. S2), which are considered to be Mg-ACCs (amorphous MgCaCO3) (Fig. 2, Movies S4 and S5)". Figure S2 which the authors refer to shows only broken sections of organic sheath at different stages of mineralization. Movie S4 shows that only in a few regions some vesicles exhibit red autofluorescence interpreted as Mg-ACC (S5 is missing but probably the authors were referring to S3). In their previous paper (Dubicka et al 2023: Heliyon), the authors used exactly the same methodology to suggest that these are intracellularly formed Mg-rich amorphous calcium carbonate particles that transform into a stable mineral phase in rotaliid Aphistegina lessonii. However, in Figure 1D (Dubicka et al 2023) the apparently carbonate-loaded vesicles show the same red autofluorescence as the test, whereas in their current paper, no evidence of autofluorescence of Mg-ACC grains accumulated within the "gel-like" organic matrix is given. The S3 and S4 movies show circulation of various fluorescing components, but no initial phase of test formation is observable (numerous mineral grains embedded within the organic matrix - Figures 3A and B - should be clearly observed also as autofluorescence of the whole layer). Thus the crucial argument supporting the calcification model (Figure 5) is missing. There is no support for the following interpretation (lines 199-203) "The existence of intracellular, vesicular intermediate amorphous phase (Mg-ACC pools), which supply successive doses of carbonate material to shell production, was supported by autofluorescence (excitation at 405 nm; Fig. 2; Movies S3 and S4; see Dubicka et al., 2023) and a high content of Ca and Mg quantified from the area of cytoplasm by SEM-EDS analysis (Fig. S6)."</li>
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Reply: We used laser line 405nm and multiphoton excitation to detect ACCs. These wavelengths (partly) permeate the shell to excite ACCs autofluorescence. The autofluorescence of the shells is present as well, but it is not clearly visible in movieS4 as the fluorescence of ACCs is stronger. This may be related to the plane/section of the cell which is shown. The laser permeates the shell above the ACCs (short distance), but to excite the shell CaCO3 around foraminifera in the same three-dimensional section where ACCs are shown, the light must pass a thick CaCO3 area due to the three-dimensional structure of the foraminifera shell. Therefore, the laser light intensity is reduced. In a revised version a movie/image with reduced threshold will be shown.
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<ol>
<li>The authors suggest that "no organic matter was detected between the needles of the porcelain structures (Figures 3E; 3E; S4C, and S5A)". Such a suggestion, which is highly unusual considering that biogenic minerals almost by definition contain various organic components, was made based only on FE-SEM observation. The authors should either provide clearcut evidence of the lack of organic matter (unlikely) or may suggest that intense calcium carbonate precipitation within organic matrix gel ultimately results in a decrease of the amount of the organic phase (but not its complete elimination), alike the pure calcium carbonate crystals are separated from the remaining liquid with impurities ("mother liquor"). On the other hand, if (249-250) "organic matrix involved in the biomineralization of foraminiferal shells may contain collagen-like networks", such "laminar" organization of the organic matrix may partly explain the arrangement of carbonate fibers parallel to the surface as observed in Fig. 3E1.</li>
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Reply: We agree with the reviewer that biogenic minerals should, by definition, contain some organic components. We wrote that "no organic matter was detected between the needles of the porcelain structures” as we did not detect any organic structures based only on our FE-SEM observations. We are convinced that the shell incorporates a limited amount of organic matrix. We will rephrase this part of the text to avoid further confusion.
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<ol>
<li>The author's observations indeed do not show the formation of individual skeletal crystallites within intracellular vesicles, however, do not explain either what is the structure of individual skeletal crystallites and how they are formed. Especially, what are the structures observed in polarized light (and interpreted as calcite crystallites) by De Nooijer et al. 2009? The author's explanation of the process (lines 213-216) is not particularly convincing "we suspect that the OM was removed from the test wall and recycled by the cell itself".</li>
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Reply: Thank you for this comment. We will do our best to supplement our explanations. We are aware of the structures observed in polarized light by De Nooijer et al. (2009). However, Goleń et al. (2022, Protist, <a href="https://doi.org/10.1016/j.protis.2022.125886">https://doi.org/10.1016/j.protis.2022.125886</a>) showed that organic polymers may also exhibit light polarization. Additional experimental studies are needed to distinguish these types of polarization. We will aim to investigate this issue in our future research.
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<ol>
<li>The following passage (lines 296-304) which deals with the concept of mesocrystals is not supported by the authors' methodology or observations. The authors state that miliolid needles "assembled with calcite nanoparticles, are unique examples of biogenic mesocrystals (see Cölfen and Antonietti, 2005), forming distinct geometric shapes limited by planar crystalline faces" (later in the same passage the authors say that "mesocrystals are common biogenic components in the skeletons of marine organisms" (are they thus unique or are they common)? It is my suggestion to completely eliminate this concept here until various crystallographic details of the miliolid test formation are well documented.</li>
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Reply: Our intention was to express that mesocrystals are common biogenic components in the skeletons of marine organisms, however Miliolid needles that form distinct geometric shapes limited by planar crystalline faces are unique type of mesocrystals.

Meike Stumpp

Claude Desplan

eLife assessment
This manuscript provides important information on the calcification process, especially the properties and formation of freshly formed tests (the foraminiferan shells), in the miliolid foraminiferan species Pseudolachlanella eburnea. The evidence from the high-quality SEM images is solid, but the evidence is incomplete when it comes to the specificity of the auto-fluorescent signals for calcified structures, or the presence of photosynthetic (living) symbionts, which are not verified experimentally. The conclusions based on fluorescent imagery therefore do not have strong support.

anonymous

Reviewer #1 (Public Review):
Summary: 
The manuscript by Dubicka and co-workers on calcification in miliolid foraminifera presents an interesting piece of work. The study uses confocal and electron microscopy to show that the traditional picture of calcification in porcelaneous foraminifera is incorrect.
Strengths: 
The authors present high-quality images and an original approach to a relatively solid (so I thought) model of calcification.
Weaknesses: 
There are several major shortcomings. Despite the interesting subject and the wonderful images, the conclusions of this manuscript are simply not supported at all by the results. The fluorescent images may not have any relation to the process of calcification and should therefore not be part of this manuscript. The SEM images, however, do point to an outdated idea of miliolid calcification. I think the manuscript would be much stronger with the focus on the SEM images and with the speculation of the physiological processes greatly reduced.

Reviewer #2 (Public Review):
Summary: 
Dubicka et al. in their paper entitled " Biocalcification in porcelaneous foraminifera" suggest that in contrast to the traditionally claimed two different modes of test calcification by rotallid and porcelaneous miliolid formaminifera, both groups produce calcareous tests via the intravesicular mineral precursors (Mg-rich amorphous calcium carbonate). These precursors are proposed to be supplied by endocytosed seawater and deposited in situ as mesocrystals formed at the site of new wall formation within the organic matrix. The authors did not observe the calcification of the needles within the transported vesicles, which challenges the previous model of miliolid mineralization. Although the authors argue that these two groups of foraminifera utilize the same calcification mechanism, they also suggest that these calcification pathways evolved independently in the Paleozoic.
Strengths: 
The authors document various unknown aspects of calcification of Pseudolachlanella eburnea and elucidate some poorly explained phenomena (e.g., translucent properties of the freshly formed test) however there are several problematic observations/interpretations which in my opinion should be carefully addressed.
Weaknesses: 
1. The authors (line 122) suggest that "characteristic autofluorescence indicates the carbonate content of the vesicles (Fig. S2), which are considered to be Mg-ACCs (amorphous MgCaCO3) (Fig. 2, Movies S4 and S5)". Figure S2 which the authors refer to shows only broken sections of organic sheath at different stages of mineralization. Movie S4 shows that only in a few regions some vesicles exhibit red autofluorescence interpreted as Mg-ACC (S5 is missing but probably the authors were referring to S3). In their previous paper (Dubicka et al 2023: Heliyon), the authors used exactly the same methodology to suggest that these are intracellularly formed Mg-rich amorphous calcium carbonate particles that transform into a stable mineral phase in rotaliid Aphistegina lessonii. However, in Figure 1D (Dubicka et al 2023) the apparently carbonate-loaded vesicles show the same red autofluorescence as the test, whereas in their current paper, no evidence of autofluorescence of Mg-ACC grains accumulated within the "gel-like" organic matrix is given. The S3 and S4 movies show circulation of various fluorescing components, but no initial phase of test formation is observable (numerous mineral grains embedded within the organic matrix - Figures 3A and B - should be clearly observed also as autofluorescence of the whole layer). Thus the crucial argument supporting the calcification model (Figure 5) is missing. There is no support for the following interpretation (lines 199-203) "The existence of intracellular, vesicular intermediate amorphous phase (Mg-ACC pools), which supply successive doses of carbonate material to shell production, was supported by autofluorescence (excitation at 405 nm; Fig. 2; Movies S3 and S4; see Dubicka et al., 2023) and a high content of Ca and Mg quantified from the area of cytoplasm by SEM-EDS analysis (Fig. S6)."
2. The authors suggest that "no organic matter was detected between the needles of the porcelain structures (Figures 3E; 3E; S4C, and S5A)". Such a suggestion, which is highly unusual considering that biogenic minerals almost by definition contain various organic components, was made based only on FE-SEM observation. The authors should either provide clearcut evidence of the lack of organic matter (unlikely) or may suggest that intense calcium carbonate precipitation within organic matrix gel ultimately results in a decrease of the amount of the organic phase (but not its complete elimination), alike the pure calcium carbonate crystals are separated from the remaining liquid with impurities ("mother liquor"). On the other hand, if (249-250) "organic matrix involved in the biomineralization of foraminiferal shells may contain collagen-like networks", such "laminar" organization of the organic matrix may partly explain the arrangement of carbonate fibers parallel to the surface as observed in Fig. 3E1.
3. The author's observations indeed do not show the formation of individual skeletal crystallites within intracellular vesicles, however, do not explain either what is the structure of individual skeletal crystallites and how they are formed. Especially, what are the structures observed in polarized light (and interpreted as calcite crystallites) by De Nooijer et al. 2009? The author's explanation of the process (lines 213-216) is not particularly convincing "we suspect that the OM was removed from the test wall and recycled by the cell itself".
4. The following passage (lines 296-304) which deals with the concept of mesocrystals is not supported by the authors' methodology or observations. The authors state that miliolid needles "assembled with calcite nanoparticles, are unique examples of biogenic mesocrystals (see Cölfen and Antonietti, 2005), forming distinct geometric shapes limited by planar crystalline faces" (later in the same passage the authors say that "mesocrystals are common biogenic components in the skeletons of marine organisms" (are they thus unique or are they common)? It is my suggestion to completely eliminate this concept here until various crystallographic details of the miliolid test formation are well documented.

Biocalcification in porcelaneous foraminifera

Figures and data

Wavelengths and dyes

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