Calcium Ions as Conjugation-Specific Regulators in Paramecium caudatum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This ms by Haga describes painstaking, potentially interesting observations on the dynamics and cyto-geography of calcium ion distribution through progressive stages of the mating/conjugation process of the ciliate, Paramecium caudatum. These observations, I acknowledge, are well worth of being published. However, they unfortunately lie completely buried in a text that is extremely bewildering on both the scientific and formal sides, and needs (to my personal opinion) in-depth rethinking, reorganization and rewriting. As it stands, the ms is practically unreadable and hard to be commented providing constructive point-to-point suggestions/criticisms. I can outline only a few of the major problems and misconceptions affecting it rather negatively.

(1) The ms title emphasizes the realization of a calcium “atlas” in mating paramecia. An atlas is commonly accepted to be ‘a bound collection of tables, charts, or plates’, and this denomination does not seem to properly apply to the experimental context of the ms.

(2) The Introduction opens with themes (“eukaryotic classification”, “Paramecium potential to become a model organism for understanding the origin and evolution of sex”, and “focus on genetic information related to sex differentiation and complementary sex recognition”) that have nothing to do with the experimental (calcium) analyses of the ms.

(3) That “Paramecium reproduces sexually” is a wrong concept anticipated in the Introduction and reiterated throughout the text. Paramecia (and ciliates/unicellular eukaryotes in general) reproduce (duplicate/multiply) only asexually by binary fission. It is a fact that two paramecia start mating and the same two paramecia stop mating. Sexual reproduction, that leads to the development of a new zygote/individual, is an evolutionary acquisition of the multicellular life. On this topic, I would suggest the Author to consult the milestone 1980 textbook by D. Nanney “Experimental Ciliatology”, as well as “Cap 4, Reproduction and Sex” in 1986 textbook “Protozoa and Other Protists” by M. Sleigh.

(4) Starting from the last paragraph of the Introduction it is emphasized that the ms reports (i) “the first comprehensive visualization/atlas of temporally and spatially regulated Ca2+ signals associated with the sexual cycle of Paramecium, (ii) “an unexpected discovery … of the formation of microdomains between three calcium ion accumulation centers, and …. (iii) novel insights into the role of Ca2+ as a central regulatory signal in sexual reproduction and the conservation of calcium signaling mechanisms.” These three inferences are hard to be accepted considering that the mere visualization of spatio-temporal variations of sites of calcium ion concentration in the mating paramecia does not necessarily involve functional and conservation significance. It would be useful/necessary to integrate the figures of the progressive cyto-geographic variations of the calcium ion concentrations with diagrams that make the reader able to promptly associate these variations with specific (nuclear, cytoplasmic or cortical) developmental processes/phenomena of the mating cell. (With regard to the figures, let me add that they are presented in a very confused order and scarce attention to the relevant indications. A first example relates to figure 2. Why “A photograph of the regional distribution of calcium fluorescence signals in conjugating Paramecium, measured 360 minutes after the onset [of what?]” anticipates photographs taken from cells involved in earlier stages of mating? Which is the contextual meaning of indicating with a white arrow “a calcium fluorescence particle within the extracellularly exposed alveolar sac”? How can an alveolar sac (a sub-plasma membrane pellicular structure) be extracellularly exposed? A second example relates to figure 4. How is it possible that “a cell in the mating reaction displayed the formation of approximately 20 fluorescent food vacuoles” and “In contrast, the cell during the dividing phase did not exhibit any food vacuoles”? To my knowledge a paramecium may divide if it eats, and may mate if it is deprived of food.

(5) Why including the subsection “The method for the isolation of mating-competent cells” into the Results section and not into the Material and Methods section? And why in the associated figure 1 the green cell depicted as paired with the red cell (see the ring lacking the designation #6) then disappears (see rings from 7 to 10) despite the legend says that the two paired cells “stayed in place briefly, allowing them to be isolated with a micropipette”?

(6) The Discussion starts emphasizing that the finding that “calcium ions … are involved in a series of sex-specific events throughout the conjugation process … provides an important experimental system for estimating the evolutionary origin of sexual reproduction.” This conclusion seems to me to be an absolutely gratuitous claim, because it implies a functional equivalence of the ciliate conjugation with the binary sex of animals. Sex differentiation in ciliates is a property specific of the migratory/male and stationary/female gamete-nuclei, not of the mating cells. The mating type systems of ciliates are not solely of a binary type, as in most Paramecium species. Much more frequently they are of a high-multiple (open) type as it is the case in hypotrichs, and fertile mating pairs may equally be formed between cells that are genetically different as well as between cells that are genetically identical.  A second part of the Discussion then swifts to tentatively explain the observed variations of “the calcium ion fluorescent signal at the cell surface” with a key role played by “region-specific micro-domains of the alveolar sac”. Lacking any schematic representation of the alveolar sacs as basic structural and functional components of the ciliate cortex, I strongly doubt that any reader who is not familiar with Paramecium anatomy can grasp something from the proposed explanation. Furthermore, in the Results section these structures have only parenthetically been mentioned in subsection 3.5 (“Areas with increased fluorescence were located in the cortical region, forming the cell surface and the alveolar sac (a flattened sac structure between the cell membrane and the microtubule network, unique to this supergroup, giving the Alveolata its name)”). The final part of Discussion, bringing onto the scene “The Last Common Eukaryotic Ancestor (LECA) …” and “The fusion gene of calmodulin and kinase genes [that] can serve as an indicator of ongoing evolution from prokaryotes to eukaryotes”, seems to me to frankly exceed in fantasy.

This ms by Haga describes painstaking, potentially interesting observations on the dynamics and cyto-geography of calcium ion distribution through progressive stages of the mating/conjugation process of the ciliate, Paramecium caudatum. These observations, I acknowledge, are well worth of being published. However, they unfortunately lie completely buried in a text that is extremely bewildering on both the scientific and formal sides, and needs (to my personal opinion) in-depth rethinking, reorganization and rewriting. As it stands, the ms is practically unreadable and hard to be commented providing constructive point-to-point suggestions/criticisms. I can outline only a few of the major problems and misconceptions affecting it rather negatively.

(1) The ms title emphasizes the realization of a calcium “atlas” in mating paramecia. An atlas is commonly accepted to be ‘a bound collection of tables, charts, or plates’, and this denomination does not seem to properly apply to the experimental context of the ms.

(2) The Introduction opens with themes (“eukaryotic classification”, “Paramecium potential to become a model organism for understanding the origin and evolution of sex”, and “focus on genetic information related to sex differentiation and complementary sex recognition”) that have nothing to do with the experimental (calcium) analyses of the ms.

(3) That “Paramecium reproduces sexually” is a wrong concept anticipated in the Introduction and reiterated throughout the text. Paramecia (and ciliates/unicellular eukaryotes in general) reproduce (duplicate/multiply) only asexually by binary fission. It is a fact that two paramecia start mating and the same two paramecia stop mating. Sexual reproduction, that leads to the development of a new zygote/individual, is an evolutionary acquisition of the multicellular life. On this topic, I would suggest the Author to consult the milestone 1980 textbook by D. Nanney “Experimental Ciliatology”, as well as “Cap 4, Reproduction and Sex” in 1986 textbook “Protozoa and Other Protists” by M. Sleigh.

(4) Starting from the last paragraph of the Introduction it is emphasized that the ms reports (i) “the first comprehensive visualization/atlas of temporally and spatially regulated Ca2+ signals associated with the sexual cycle of Paramecium, (ii) “an unexpected discovery … of the formation of microdomains between three calcium ion accumulation centers, and …. (iii) novel insights into the role of Ca2+ as a central regulatory signal in sexual reproduction and the conservation of calcium signaling mechanisms.” These three inferences are hard to be accepted considering that the mere visualization of spatio-temporal variations of sites of calcium ion concentration in the mating paramecia does not necessarily involve functional and conservation significance. It would be useful/necessary to integrate the figures of the progressive cyto-geographic variations of the calcium ion concentrations with diagrams that make the reader able to promptly associate these variations with specific (nuclear, cytoplasmic or cortical) developmental processes/phenomena of the mating cell. (With regard to the figures, let me add that they are presented in a very confused order and scarce attention to the relevant indications. A first example relates to figure 2. Why “A photograph of the regional distribution of calcium fluorescence signals in conjugating Paramecium, measured 360 minutes after the onset [of what?]” anticipates photographs taken from cells involved in earlier stages of mating? Which is the contextual meaning of indicating with a white arrow “a calcium fluorescence particle within the extracellularly exposed alveolar sac”? How can an alveolar sac (a sub-plasma membrane pellicular structure) be extracellularly exposed? A second example relates to figure 4. How is it possible that “a cell in the mating reaction displayed the formation of approximately 20 fluorescent food vacuoles” and “In contrast, the cell during the dividing phase did not exhibit any food vacuoles”? To my knowledge a paramecium may divide if it eats, and may mate if it is deprived of food.

(5) Why including the subsection “The method for the isolation of mating-competent cells” into the Results section and not into the Material and Methods section? And why in the associated figure 1 the green cell depicted as paired with the red cell (see the ring lacking the designation #6) then disappears (see rings from 7 to 10) despite the legend says that the two paired cells “stayed in place briefly, allowing them to be isolated with a micropipette”?

(6) The Discussion starts emphasizing that the finding that “calcium ions … are involved in a series of sex-specific events throughout the conjugation process … provides an important experimental system for estimating the evolutionary origin of sexual reproduction.” This conclusion seems to me to be an absolutely gratuitous claim, because it implies a functional equivalence of the ciliate conjugation with the binary sex of animals. Sex differentiation in ciliates is a property specific of the migratory/male and stationary/female gamete-nuclei, not of the mating cells. The mating type systems of ciliates are not solely of a binary type, as in most Paramecium species. Much more frequently they are of a high-multiple (open) type as it is the case in hypotrichs, and fertile mating pairs may equally be formed between cells that are genetically different as well as between cells that are genetically identical.  A second part of the Discussion then swifts to tentatively explain the observed variations of “the calcium ion fluorescent signal at the cell surface” with a key role played by “region-specific micro-domains of the alveolar sac”. Lacking any schematic representation of the alveolar sacs as basic structural and functional components of the ciliate cortex, I strongly doubt that any reader who is not familiar with Paramecium anatomy can grasp something from the proposed explanation. Furthermore, in the Results section these structures have only parenthetically been mentioned in subsection 3.5 (“Areas with increased fluorescence were located in the cortical region, forming the cell surface and the alveolar sac (a flattened sac structure between the cell membrane and the microtubule network, unique to this supergroup, giving the Alveolata its name)”). The final part of Discussion, bringing onto the scene “The Last Common Eukaryotic Ancestor (LECA) …” and “The fusion gene of calmodulin and kinase genes [that] can serve as an indicator of ongoing evolution from prokaryotes to eukaryotes”, seems to me to frankly exceed in fantasy.

Author Response

We would like to thank Reviewer 1 for his many valuable comments. We will describe our response and thoughts on each comment below.

(1) The ms title emphasizes the realization of a calcium “atlas” in mating paramecia. An atlas is commonly accepted to be ‘a bound collection of tables, charts, or plates’, and this denomination does not seem to properly apply to the experimental context of the ms.

Response 1.

As you pointed out, the term "Atlas" is used to mean an atlas, but it is also used metaphorically to describe various topographies. In this paper, we used it to illustrate the calcium signal that occurs throughout the entire mating cells of Paramecium. We identified four regions along the long axis of the Paramecium cell body as the central areas of the calcium signal and compared the concentration changes to a topographical map. Since the calcium signal fluorescence image was shown to be a four-dimensional phenomenon, we used the term "atlas" to describe it concisely. Four regions were selected for statistical analysis of changes in fluorescence intensity. These regions are based on a topographical map that can be represented by coordinate axes.

 

(2) The Introduction opens with themes (“eukaryotic classification”, “Paramecium potential to become a model organism for understanding the origin and evolution of sex”, and “focus on genetic information related to sex differentiation and complementary sex recognition”) that have nothing to do with the experimental (calcium) analyses of the ms.

Response 2

The main finding of this paper is the description of the characteristics of calcium signals that occur specifically during mating. Since the mating substance is a calcium-dependent protein kinase, this work focuses on the role of calcium in the mating process. One of the most important features of eukaryotes is their ability to reproduce sexually. Although it has been argued that the definition of sexual reproduction varies depending on an organism's lifestyle, this paper will focus on the basic form of meiosis, which produces a germ nucleus and fuses it with the germ nucleus of a different individual to form the offspring's genome. This paper provides a broad overview of the research background aligned with the theme of this special issue, which emphasizes the phylogenetic trees of eukaryotic supergroups.

 

(3) That “Paramecium reproduces sexually” is a wrong concept anticipated in the Introduction and reiterated throughout the text. Paramecia (and ciliates/unicellular eukaryotes in general) reproduce (duplicate/multiply) only asexually by binary fission. It is a fact that two paramecia start mating and the same two paramecia stop mating. Sexual reproduction, that leads to the development of a new zygote/individual, is an evolutionary acquisition of the multicellular life. On this topic, I would suggest the Author to consult the milestone 1980 textbook by D. Nanney “Experimental Ciliatology”, as well as “Cap 4, Reproduction and Sex” in 1986 textbook “Protozoa and Other Protists” by M. Sleigh.

Response 3.

I purchased and studied Nanney’s book shortly after it was published, and I was inspired by its content, which highlighted the uniqueness of ciliate cells and the critical perspectives they offer. The conjugation process in Paramecium species involves meiosis of the micronucleus. The resulting germ nucleus, called a pronucleus, fuses with a pronucleus from a partner of mating to form a synkaryon. From this, four macronuclei and one micronucleus are produced, completing the genome organization for the next generation.  Based on this process, the conjugation in Paramecium is considered a form of sexual reproduction. I discussed this point with Dr. Nanney at the Ciliate Molecular Biology Conference held in the U.S. in the 1980s. We agreed that the conjugation process in Paramecium does not violate the criteria for sexual reproduction. The number of macronuclear anlagen assesses the number of offspring produced. In Paramecium caudatum, four macronuclear anlagen are produced in each mating cell and distributed among the four cells resulting from two cell divisions of the exconjugant. Therefore, eight offspring are produced from one mating pair. This forms the basis for our use of the term reproduction. Macronuclear genome editing occurs in each karyonide, increasing diversity in the progeny.

 

(4) Starting from the last paragraph of the Introduction it is emphasized that the ms reports (i) “the first comprehensive visualization/atlas of temporally and spatially regulated Ca2+ signals associated with the sexual cycle of Paramecium, (ii) “an unexpected discovery … of the formation of microdomains between three calcium ion accumulation centers, and …. (iii) novel insights into the role of Ca2+ as a central regulatory signal in sexual reproduction and the conservation of calcium signaling mechanisms.” These three inferences are hard to be accepted considering that the mere visualization of spatio-temporal variations of sites of calcium ion concentration in the mating paramecia does not necessarily involve functional and conservation significance. It would be useful/necessary to integrate the figures of the progressive cyto-geographic variations of the calcium ion concentrations with diagrams that make the reader able to promptly associate these variations with specific (nuclear, cytoplasmic or cortical) developmental processes/phenomena of the mating cell. (With regard to the figures, let me add that they are presented in a very confused order and scarce attention to the relevant indications.

Response 4.

Based on these suggestions, we have combined and improved the figures and added a new table to help the reader quickly identify specific mating-cell processes (nucleus, cytoplasm, or cortex). Revised Figure 3 shows changes in the fluorescence signal over time after mating begins, starting from cells in the vegetative growth phase before conjugation starts. The time course of fluorescence signal intensity across the four regions was compared, and statistical significance tests were performed between areas and at each time point; the results were displayed as a heat map. The revised Figure 4 displays data comparing cells at 1 and 6 hours after mating. Notable increases were observed in the anterior and posterior regions at 1 and 6 hours, respectively, indicating that the rises in calcium signaling are driven by an active mechanism rather than passive diffusion. In revised Figure 5, the calcium ion threshold for maintaining mating reactivity and mating pair formation is shown. Extracellular and intracellular calcium ion concentrations exert a switching effect on the progression of mating. Revised Figure 6 illustrates the distribution pattern and shape changes of the fluorescent particles within the conjugate. An increase in particle size was observed after 5 and 6 hours of conjugation. The quantitative aspect of this change is a subject for future research and is not covered in this paper. The newly attached Table 1 presents the measured calcium ion concentration thresholds in organelles and various cell parts throughout the conjugation process. It was found that the threshold increased by more than 5 times within 3 hours. However, this study provides no information that allows us to comment on the chemical reaction system underlying the calcium ion threshold.

 

A first example relates to figure 2. Why “A photograph of the regional distribution of calcium fluorescence signals in conjugating Paramecium, measured 360 minutes after the onset [of what?]” anticipates photographs taken from cells involved in earlier stages of mating? Which is the contextual meaning of indicating with a white arrow “a calcium fluorescence particle within the extracellularly exposed alveolar sac”? How can an alveolar sac (a sub-plasma membrane pellicular structure) be extracellularly exposed? A second example relates to figure 4.

Response

In response to this comment, we have reorganized the data chronologically, as shown in Figures 3, 4, and 6. In Paramecium, the mechanism by which the alveolar sac is exposed to the outside of the cell while still enclosed by a membrane is unknown, but this phenomenon is often observed during the drying process on glass slides.

 

How is it possible that “a cell in the mating reaction displayed the formation of approximately 20 fluorescent food vacuoles” and “In contrast, the cell during the dividing phase did not exhibit any food vacuoles”? To my knowledge a paramecium may divide if it eats, and may mate if it is deprived of food.

Response

The caption for this section shows the appearance of calcium signals in cells with numerous food vacuoles and in dividing cells without food vacuoles. This indicates that the calcium ion concentration on the cell surface does not increase in either proliferating cells with many phagosomes or dividing cells, supporting the finding that the increase in calcium ions is specific to the mating process.

 

(5) Why including the subsection “The method for the isolation of mating-competent cells” into the Results section and not into the Material and Methods section? And why in the associated figure 1 the green cell depicted as paired with the red cell (see the ring lacking the designation #6) then disappears (see rings from 7 to 10) despite the legend says that the two paired cells “stayed in place briefly, allowing them to be isolated with a micropipette”?

Response 5.

This is because this is a new method established for the first time in this experiment. Previously, it was thought that cell aggregates formed during mating were synchronously committed to the mating process. However, this study revealed that the proportion of cells entering commitment was time-dependent, peaking approximately 30 minutes after the start of the mating reaction.

The figure legend has been revised to better describe the experiment's progress. Only wild-type cells were used for observation, so mutants were excluded. Fluorescence microscopy was performed on individual cells, and the figures are uniformly colored red (wild type). Green cells were also observed, but no significant difference was detected.

 

(6) The Discussion starts emphasizing that the finding that “calcium ions … are involved in a series of sex-specific events throughout the conjugation process … provides an important experimental system for estimating the evolutionary origin of sexual reproduction.” This conclusion seems to me to be an absolutely gratuitous claim, because it implies a functional equivalence of the ciliate conjugation with the binary sex of animals. Sex differentiation in ciliates is a property specific of the migratory/male and stationary/female gamete-nuclei, not of the mating cells.

Response 6.

This appears to be an entirely baseless claim, as it implies a functional equivalence between ciliate fusion and the dual sex in animals. The process of sexual reproduction is common in multicellular organisms and involves the formation of heterogametes and the joining of gametes from different individuals. However, during the early stages of eukaryotic cell evolution from bacterial cells, it was believed that sexual reproduction occurred at the level of the germ nucleus.

 

The mating type systems of ciliates are not solely of a binary type, as in most Paramecium species. Much more frequently, they are of a high-multiple (open) type, as is the case in hypotrichs, and fertile mating pairs may equally be formed between cells that are genetically different as well as between cells that are genetically identical.  A second part of the Discussion then swifts to tentatively explain the observed variations of “the calcium ion fluorescent signal at the cell surface” with a key role played by “region-specific micro-domains of the alveolar sac”. Lacking any schematic representation of the alveolar sacs as basic structural and functional components of the ciliate cortex, I strongly doubt that any reader who is not familiar with Paramecium anatomy can grasp something from the proposed explanation. Furthermore, in the Results section these structures have only parenthetically been mentioned in subsection 3.5 (“Areas with increased fluorescence were located in the cortical region, forming the cell surface and the alveolar sac (a flattened sac structure between the cell membrane and the microtubule network, unique to this supergroup, giving the Alveolata its name)”).

Response

The revised version includes an electron micrograph of the surface structures of Paramecium (Figure 6, Stelly 1991), showing alveoli to help readers better understand these structures.

 

The final part of Discussion, bringing onto the scene “The Last Common Eukaryotic Ancestor (LECA) …” and “The fusion gene of calmodulin and kinase genes [that] can serve as an indicator of ongoing evolution from prokaryotes to eukaryotes”, seems to me to frankly exceed in fantasy.

 

Response.

With the recent accumulation of molecular biological data, LECA has become a testable hypothesis. In relation to calcium and cellular function, the binding state of calcium ions with specific amino acids has been examined at the level of the calcium-binding protein's three-dimensional structure. Additionally, the existence of fusion genes involving calcium-binding protein genes and kinase genes has also been considered at the kingdom level.

Assuming LECA, we deduced the process by which unicellular eukaryotic cells evolved from bacterial cells using molecular biological data. We also assessed the usefulness of a dataset that included calcium-activated protein kinase genes. This was presented as a suggestion about research perspectives and possibilities. This paper was submitted to a special issue of MDPI to gain a deeper understanding of the evolutionary tree, including unicellular eukaryotes, and therefore presents a new perspective essential for phylogenetic tree analysis. Generally, formulating testable hypotheses about unknown phenomena and conducting empirical experiments are well-established scientific methods. Considering dreams, hopes, and realization as part of a continuum is also an acceptable approach in individual research. We sincerely value your detailed opinions and helpful suggestions. We will continue to pursue an experimental approach based on testable hypotheses and push the boundaries of reality.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Calcium Atlas and Stage-Specific Thresholds in the Conjugation of Paramecium caudatum” investigates the intracellular localization of calcium ions in mating-competent Paramecium caudatum cells using fluorescence-based imaging. While the topic is of potential interest, the experimental approach is rather straightforward, and several important issues need to be addressed:

1. The manuscript does not clearly specify the number of independent biological replicates used for statistical analysis. Were all images acquired from a single sample preparation? If so, the reproducibility and robustness of the results are questionable. Detailed information on sample replication and data variability should be provided.
2. The influence of extracellular calcium concentration is evaluated only at the population level. To strengthen the conclusions, the authors should include data showing how reduced or elevated extracellular calcium levels affect intracellular calcium distribution patterns
3. The biological relevance of the observed calcium distribution remains unclear. Co-localization studies with key proteins or substrates are necessary to elucidate the mechanistic context.
4. The results section is difficult to follow due to the excessive number of subsections presenting similar data. For instance, the subsections in Section 3.11 all describe the effects of calcium concentration and should be merged into a single, coherent section. The data should be summarized in one unified figure containing several consistently formatted panels.
5. The data in Figure 6 should be reorganized and presented as a single consolidated graph.

Author Response

We thank the reviewer for the comments. We have revised the entire manuscript and included explanations about the statistical procedures. We have also reorganized the graphs and created a table summarizing the changes in calcium ion thresholds during the early stages of the conjugation process. We have noted the specific responses to each comment.

 

Comment 1. The manuscript does not clearly specify the number of independent biological replicates used for statistical analysis. Were all images acquired from a single sample preparation? If so, the reproducibility and robustness of the results are questionable. Detailed information on sample replication and data variability should be provided.

 

Response 1.

Figures 2, 3, and 4 all display data collected from individual cells. In Figure 5, A shows n=10, B shows n=12, and C shows n=68 for holdfast and n=62 for paroral. This information has been added to the text.

When cells committed to the mating process were prepared using the method shown in Figure 1, calcium signals of the same level were detected in 100% of the cells.

 We have confirmed that this method reliably detects fluorescence images consistently at all time points during the conjugation process. This point was added to the text.

 

Comment 2. The influence of extracellular calcium concentration is evaluated only at the population level. To strengthen the conclusions, the authors should include data showing how reduced or elevated extracellular calcium levels affect intracellular calcium distribution patterns.

 

Response 2.

In Paramecium that have not yet entered the conjugation process, the effect of changes in external calcium ion concentration on intracellular calcium distribution cannot be visualized because the intracellular calcium level does not reach the detection limit of Indo-1-AM. In the conjugation process, a weak signal is detected at the anterior part of cells where mating activity is expressed. The calcium distribution in other regions of the cells is not detected. An increase in intracellular calcium ions and changes in their distribution can be detectable only in cells that have initiated the mating reaction. The key finding of our study is that only the mating process increases calcium ion distribution. It is difficult to detect changes in intracellular calcium ion distribution when the external calcium concentration is altered under our experimental conditions.

 

Comment 3. The biological relevance of the observed calcium distribution remains unclear. Co-localization studies with key proteins or substrates are necessary to elucidate the mechanistic context.

 

Response 3.

We acknowledge that your point is central to our research goals and is highly significant. The increase in calcium ion concentration and the change in its intracellular distribution are consistent with the report that EMM, a phenomenon that always occurs before micronuclei enter meiosis during the conjugation process, can be induced in mating-reactive cells by microinjecting calcium ions. This effect occurs only in mating-reactive cells, not in mating-nonreactive cells.  However, we have not yet identified the molecule that interacts with calcium ions and induces meiosis in the micronucleus (the nucleus that provides the germline genome during conjugation). It is difficult to prepare cells suitable for biochemical materials and to assemble an experimental system for molecular-level assays. In this study, we estimated the calcium ion threshold for the progression of the conjugation process, providing a quantitative criterion for identifying target molecules for calcium ions.

 

Comment 4. The results section is difficult to follow due to the excessive number of subsections presenting similar data. For instance, the subsections in Section 3.11 all describe the effects of calcium concentration and should be merged into a single, coherent section. The data should be summarized in one unified figure containing several consistently formatted panels.

 

Response 4.

We revised Figures 3, 4, and 5 and added Figures 6, 7, 8, and 9, as well as Table 1, to improve clarity. Changes and emphasis in Figures 3, 4, and 5 are shown in the text. Figure 6 shows the changes in calcium signals over time in the mating pairs. In Table 1, we specify the calcium concentration threshold needed for the conjugation process to advance to the next stage. Figure 7 displays the characteristics and thresholds of the calcium atlas and summarizes them to guide future research strategies. In Figure 8, we provide information to help readers understand the structure of the alveolar sac by citing electron microscopy. In Figure 9, we show a photograph of the alveolar sac on the cell membrane's outer surface, with fluorescent particles visible.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Please find the attachment.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscipt significantly improved, it can be suitable for publication in current form.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf