Microalgal Lipid Profile and Their Dietary Impact on Drosophila melanogaster
Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for Authors
< !--StartFragment-->
Submitted study is devoted to the fatty acid composition of four species of green freshwater algae and their dietary effect on Drosophila melanogaster. It was concluded that dietary supplementation of micro algae biomass extended median lifespan mainly in males. However, lifespan extension did not correlate with lipids content and fatty acid composition of dietary algae. In spite of this, the authors concluded that micro algae lipids quality may be critical for modulating health outcomes. This conclusion contradicts the results obtained.
In addition, I could not agree with the conclusion of the authors that “proportion of fatty acids differed significantly between species “, because their fatty acid profiles are very similar.
I believe, that the manuscript contains significant errors in the lipids and fatty acid analysis.
It is well-known that fatty acid compositions of algae depend on their taxonomic position. Different classes of algae are characterized by different fatty acid profiles.
In manuscript, four species of microalgae belonging to two classes were studied. Among them, Nannochloropsis limnetica from class Eustigmatophyceae is a green microalga known for producing high level of PUFAs particularly 20:5n-3 (20-30% of total fatty acids). See for example, Krienitz and Wirth, Science. Reports 2006). Also, in a previous paper by Yuchen Li, Svitlana Miros et al.), it was noticed “Species belonging to the Nannochloropsis genus are generally rich in 20:5n-3”. However, this fatty acid was not found in the species, but 18:1n-9 was predominant.
Another three species belonging to class Chlorophycea are known to characterize by a high proportion of PUFAs, particularly 18:3n-3 (up to 50%), 18:2n-6 (20%) and 16:0, whereas 18:1n-9 is a minor component. However, in all studied samples of algae 18:1n-9 was the main and it accounted for more than 50% of the total fatty acids. But specific for green algae 18:3n-3 and C16 PUFAs were not found.
So, fatty acid profiles of the studied species differed significantly from that of reported earlier.
The main problem of this study is methodological errors. The methodological errors likely led to distorted results.
The authors wrote that lipids were extracted by the method of Bligh and Dyer (1959). Really, the lipid extraction method by Bligh and Dyer (1959) is one of the standard procedures to isolate total lipids from biological samples. It based on solvent system consisting of chloroform/methanol, 2:1 (v/v). However the authors used the solvent ratio 4:5. Moreover, only 1 ml of this solvent mixture was used. It is too small volume for extraction.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for Authors
Manuscript ID: 4123949
Title: Microalgal Lipid Profile and Their Dietary Impact on Drosophila melanogaster.
Authors: Svitlana Miros, Svitlana Bilokon, Yiting Han and Ronald Halim.
General Comments to the Authors
The submitted study investigates the effect of microalgae supplementation in the diet of the fruit fly Drosophila melanogaster on its lifespan and other growth characteristics. The authors focus on the fatty acid (FA) composition of the microalgae. However, this aspect represents the key problem in the work, as the data point to the incorrectness of the performed FA analysis.
First, the HP-5 column used is not a standard or optimal choice for routine fatty acid analysis, especially for their accurate identification in mixtures. While this approach is technically possible and acceptable for a general characterization of the FA profile, the obtained profile raises serious concerns. It is unusually impoverished and contradicts numerous literature data. The FA profiles obtained by the authors include too little diversity of fatty acids.
For instance, the FA profiles of Chlorella are very well studied (e.g., Petkov and Garcia, 2007 “Which are fatty acids of the green alga Chlorella?”; Prokopkin et al., 2021 “Assessing the Reliability of Quantitative Fatty Acid Signature Analysis and Compound-Specific Isotope Analysis-Based Mixing Models for Trophic Studies”). Like most green algae representatives, they contain not only 18:2n-6 but also 18:3n-3 and C16 PUFAs. The authors did not detect these acids at all. In Nannochloris limnetica (Eustigmatophyceae, but not green algae), the authors also failed to detect fatty acids characteristic of these algae, namely 20:5n-3 and 20:4n-6 (e.g., Krienitz and Wirth, 2006 “The high content of polyunsaturated fatty acids in Nannochloropsis limnetica (Eustigmatophyceae) and its implication for food web interactions, freshwater aquaculture and biotechnology.”). This simplification of the profile may indicate incorrect FA analysis or specific growth conditions. However, according to the description of the algae cultivation and growth conditions, they were optimal.
Furthermore, the authors found a huge discrepancy between the gravimetrically determined total lipid content and the sum of fatty acids. While non-saponifiable lipids (pigments, sterols) contribute to the gravimetric analysis, and losses are possible during the GC sample preparation stage, some discrepancies could indeed occur (but even these should have been commented on by the authors in the Discussion). However, a difference of 7-14 times between total lipids and the sum of fatty acids (FAs) falls far outside the standard explanation of "non-saponifiable lipids and losses." For example, if the sum of FAs in lipids is 14 times smaller, it means that fatty acids constitute less than 7% of total lipids, which contradicts the biochemistry and physiology of algae.
The presented results show a substantial and systematic discrepancy between the gravimetrically determined total lipid content and the sum of fatty acids determined by GC-FID. The difference factor ranges from 7 to 14 times depending on the strain. Such a significant difference cannot be explained only by the contribution of the non-saponifiable fraction and suggests either a methodological artifact or the need for additional clarification.
Considering all of the above, there is a well-founded assumption that the analysis methodology used (transesterification and GC on a non-polar HP-5 column) could have led not only to quantitative losses but also to qualitative distortions of the profile.
Specific Comments for Improvement
Lines 43-44: The phrase "PUFAs derived from microalgae contribute to human health in multiple ways..." is incorrect and needs revision. Firstly, it is not only PUFAs themselves but also, to a large extent, derivatives from specific PUFAs (not all) that affect human health. This needs clarification. Secondly, the source of PUFAs is not important.
Line 49: Please clarify how the reactor configuration affects the FA composition of microalgal biomass.
Lines 50-52: In algae, not only the percentage content differs, but the overall FA composition is also different among different algae. There are fatty acids found only in certain algal groups.
Lines 55-66: This is all correct, and the fruit fly is used as a model organism in medicine for studying a number of human diseases. However, the fatty acid requirements of these flies differ from those of humans. For example, EPA and DHA are not essential for these flies but are essential for humans. I believe the authors should exercise caution in considering the fly as a model organism in this context.
Section 2.2: Add the volumes of hexane, potassium methoxide, and methanol with acid that were added.
Line 92: Specify how methanol was removed.
Line 99: The authors should clarify whether they indeed used 20% HCl. A reference for this protocol should be provided.
Line 113: It appears that reference 19 is cited for the entire method in this section. However, the cited article uses a different method for both extraction and methanolysis and did not employ an internal standard. Therefore, the reference seems inappropriate. In my opinion, the method from article 19 is more suitable than the one described in this manuscript. Moreover, in that article, you also analyzed algae. What prompted the change in methodology? Given the incorrectness of the obtained results, the authors should reconsider the methodology for future work.
Line 118: Please add the percentage of microalgae supplement (%) in the nutrient medium.
Section 3.1: All results in this section are incorrect.
Lines 175, 188: Is N=100 the total n, or were there 100 replicates in each group? It is correct to report this separately for each group, not as a total.
Lines 201-205: This paragraph contradicts the obtained results. According to your data, the FA composition of all algae was very similar, although this completely contradicts the literature.
Lines 233; 233-243: According to Table 1, the authors detected only one PUFA, constituting about 20% and not more than half of all FAs. What are these considerations based on? In this paragraph, the FA profile is compared to literature data incorrectly, as the authors do not mention at all that their FA profiles do not correspond to what is indicated in the literature.
Line 240: According to genetic data, N. limnetica is not a green alga.
Lines 249-255: This is a repeated description of the result, not its discussion.
Line 259: The term "Correlate" is inappropriate. The authors did not perform a correlation analysis.
Lines 310-316: It is obvious that the algae introduced additional beneficial substances, enriching the food. However, based on the work's outcomes, it is impossible to conclude what played the key role in the positive effect on the flies' lifespan.
The Conclusion needs to be revised according to the comments on the entire manuscript.
The references provided in the review should be considered only as examples supporting the reviewer's statements.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report (New Reviewer)
Comments and Suggestions for Authors
Dear authors and editors,
The manuscript Microalgal lipid profile and their dietary impact on Drosophila melanogaster presents interesting and valuable data on freshwater green microalgae which can serve as a physiologically safe dietary lipid source for D. melanogaster. Additionally, the insect itself represents a suitable in vivo model for screening the nutritional potential of microalgal lipids. The methodology is rigorous and appropriate, and the data are thoroughly analysed. The authors present clear, logically structured arguments, and their conclusions are well supported by the results. Тhe figures and tables effectively illustrate the key findings. Overall, this manuscript advances current understanding and will be of interest to researchers and practitioners in the field. I recommend acceptance for publication.
I found only one minor technical flaw: the Latin name T. obliquus in row 236 should be italicised.
Author Response
Comment 1: I found only one minor technical flaw: the Latin name T. obliquus in row 236 should be italicised.
Response 1: Thank you for pointing this out. We have corrected that.
Thank you for your time and revision.
Reviewer 4 Report (New Reviewer)
Comments and Suggestions for Authors
Overall, the manuscript "Microalgal Lipid Profile and Their Dietary Impact on Drosoph
ila melanogaster" is a very interesting experimental study. Its results are valuable for further optimising microalgal strain selection, growing conditions, and dosage in the development of nutritional supplements. Overall, the manuscript is well structured and written in clear language. The data obtained has been processed using modern statistical methods, which confirms the reliability of the results. A few comments are provided below. I hope they will help the authors improve their manuscript.
In Abstract, words such as Background, Methods, etc. can be removed.
In my opinion, the title of the manuscript needs to be clarified in terms of the microalgae used for the study. Perhaps the names of the species themselves could be added, or at least the phylum. The names of the taxa should also be added to the keywords to make it easier for researchers to find information on the species they are interested in.
Sentence "Total lipid content ranged from 17.3% to 28.1% of dry weight..." in Abstract sounds as if microalgae were grown together and their total lipids were measured. Here, it would probably be better to list the values for each species.
The introduction may include a justification for selecting these particular species for study and provide literature data on the study of their biochemical composition and impact on drosophila.
L. 265 Latin is not italicised.
Author Response
Overall, the manuscript "Microalgal Lipid Profile and Their Dietary Impact on Drosophila melanogaster" is a very interesting experimental study. Its results are valuable for further optimising microalgal strain selection, growing conditions, and dosage in the development of nutritional supplements. Overall, the manuscript is well structured and written in clear language. The data obtained has been processed using modern statistical methods, which confirms the reliability of the results. A few comments are provided below. I hope they will help the authors improve their manuscript.
Comment 1: In Abstract, words such as Background, Methods, etc. can be removed.
Response 1: Thank you for this comment. We respectfully prefer to keep the structured headings, as this format is explicitly required by the journal’s Guide for Authors. According to the MDPI journal Lipidology instructions, “Systematic reviews and original research articles should have a structured abstract… containing the following headings: Background/Objectives, Methods, Results, and Conclusions.” https://www.mdpi.com/journal/lipidology/instructions
Comment 2: In my opinion, the title of the manuscript needs to be clarified in terms of the microalgae used for the study. Perhaps the names of the species themselves could be added, or at least the phylum. The names of the taxa should also be added to the keywords to make it easier for researchers to find information on the species they are interested in.
Response 2: Thank you for your suggestion. We have added the recommended microalgae taxa names to the Keywords section. We would prefer to keep the title unchanged, as it already complies with the journal’s Guide for Authors, which states: “The title of your manuscript should be concise, specific and relevant. It should identify if the study reports (human or animal) trial data, or is a systematic review, meta-analysis or replication study.” We believe the current title meets these requirements and accurately reflects the scope of the study.
Comment 3: Sentence "Total lipid content ranged from 17.3% to 28.1% of dry weight..." in Abstract sounds as if microalgae were grown together and their total lipids were measured. Here, it would probably be better to list the values for each species.
Response 3: We agree with your comment. However, due to the abstract word limit, we were unable to include a full explanation. Instead, we have added a clarification stating that the microalgae were cultivated separately (Line 18).
Comment 4: The introduction may include a justification for selecting these particular species for study and provide literature data on the study of their biochemical composition and impact on drosophila.
Response 4: Thank you for this helpful suggestion. We have revised the Introduction to include a clear justification for the selection of the four microalgal species (L61-69).
Comment 5: L. 265 Latin is not italicised.
Response 5: Thank you for pointing this out. We have corrected that (L415).
Thank you for your time and revision. Much appreciated.
Reviewer 5 Report (New Reviewer)
Comments and Suggestions for Authors
The article is quite interesting and simple in its idea and experimentation. Additional experiments could make it more robust and could help gain some helpful insights into the properties of these microalgae as additives in the diet of Drosophila.
I suggest indicating in the Introduction or in the Discussion section what was the logic underlying the choice of the four microalgae species. Are these four among the ones listed for human consumption and is this the final aim of your study? Are they already used for animal feed production?
For an in vivo assay I would have expected positive control in addition to the negative one. Moreover, it would have been interesting to evaluate the effect of the pure fatty acid (at a reasonable concentration) in order to highlight the influence of the single component on the diet of the animals.
Was the alimentary behavior of Drosophila affected in any way from the addition of the microalgae biomass? I am thinking for example of the smell or the taste of the final product.
Lines 118-119: How was the amount of microalgae biomass chosen?
Figure 1: I suggest changing the labelling of the treatments using Ch, Sc, Nl, To (Upper- and lower-case letters), in order to standardize it. Make the two charts more homogeneous from a graphical point of view.
Lines 266-269: Indeed, why have you not analyzed other compounds of your microalgae (carotenoids, xanthophylls) so that other correlation could be made? For example some simple antioxidant tests of the biomass could have been useful to assess this property of the different species.
Author Response
The article is quite interesting and simple in its idea and experimentation. Additional experiments could make it more robust and could help gain some helpful insights into the properties of these microalgae as additives in the diet of Drosophila.
Comment1: I suggest indicating in the Introduction or in the Discussion section what was the logic underlying the choice of the four microalgae species. Are these four among the ones listed for human consumption and is this the final aim of your study? Are they already used for animal feed production?
Response 1: We thank the reviewer for this helpful suggestion. The four microalgal species were selected based on their relevance as freshwater oleaginous microalgae with documented biotechnological potential and availability from recognized culture collections. Chlorella vulgaris is already widely used in human nutrition and as a feed ingredient, whereas Tetradesmus obliquus and Scenedesmus communis are well-studied candidates for animal feed and biorefinery applications due to their robust growth and favourable biochemical composition. Nannochloropsis limnetica was included as a representative of eustigmatophytes, a group known for high lipid productivity and industrial interest but still underexplored in dietary studies using whole-organism models. We have now clarified this rationale in the Introduction section as suggested (L128-134).
Comment 2: For an in vivo assay I would have expected positive control in addition to the negative one. Moreover, it would have been interesting to evaluate the effect of the pure fatty acid (at a reasonable concentration) in order to highlight the influence of the single component on the diet of the animals.
Response 2: We agree that the inclusion of a positive control and testing of individual fatty acids would provide additional mechanistic insight into the observed biological effects. The primary aim of the present study, however, was to perform a screening assessment of whole microalgal biomass as a dietary lipid source, reflecting a realistic nutritional scenario in which organisms consume complex biomass rather than isolated lipid fractions. For this reason, we focused on comparing supplemented diets with the standard control diet.
Comment 3: Was the alimentary behavior of Drosophila affected in any way from the addition of the microalgae biomass? I am thinking for example of the smell or the taste of the final product.
Response 3: We did not directly measure feeding behavior or food intake (e.g., dye‐based consumption assays). However, several indirect observations suggest that the addition of microalgal biomass did not negatively affect food palatability or feeding activity. First, flies readily developed and survived on all supplemented media, and no avoidance behaviour or abnormal mortality was observed during the early experimental period. Second, reproductive output and body mass were maintained or improved in several treatments, which would be unlikely if food intake had been substantially reduced. Third, the supplementation level used (0.3% w/v) was relatively low and did not visibly alter the texture or consistency of the diet.
Comment 4: Lines 118-119: How was the amount of microalgae biomass chosen?
Response 4: We thank the reviewer for this question. The amount of microalgal biomass added to the diet (3 mg mL⁻¹; 0.3% w/v) was selected based on previously published studies in which microalgae supplementation was successfully applied in D.melanogaster feeding experiments without negatively affecting survival or development. We have now clarified this rationale in the revised manuscript and added the appropriate references (L190).
Comment 5: Figure 1: I suggest changing the labelling of the treatments using Ch, Sc, Nl, To (Upper- and lower-case letters), in order to standardize it. Make the two charts more homogeneous from a graphical point of view.
Response 5: Thank you for this suggestion. The labelling of the treatments has been standardized to Ch, Sc, Nl, and To, and both charts in Figure 1 have been revised to ensure a more homogeneous graphical presentation.
Comment 6: Lines 266-269: Indeed, why have you not analyzed other compounds of your microalgae (carotenoids, xanthophylls) so that other correlation could be made? For example, some simple antioxidant tests of the biomass could have been useful to assess this property of the different species.
Response 6: Thank you for this helpful suggestion. We fully agree that the evaluation of additional bioactive compounds and antioxidant capacity would strengthen the interpretation of the biological effects observed. Following the reviewer’s recommendation, we have now expanded the manuscript to include the assessment of antioxidant activity of the microalgal biomass using DPPH radical scavenging activity, FRAP assay, and total phenolic content (TPC). The corresponding results and discussion have been added to the revised manuscript.
Thank you for your time and revision. Much appreciation.
Round 2
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for Authors
In general, the arguments about the dependence of the composition of fatty acids on the conditions of cultivation and modulation of the biosynthetic pathways of L. 328-380 did not improve the manuscript, but only introduced unsubstantiated information.
“microalgae composition” L. 13 What kind of composition do you mean? Lipid or protein or elemental or pigment etc composition? Correct.
What does it mean “lipid-related components”? L. 27. Correct.
What is it “in most experimental groups”? Correct.
I do not recommend including Latin names of algae in keywords, as the lipid and fatty acid analysis of algae was carried out with serious errors.
At
least, the fragment of L. 40-52 is too far from the results of this study, so it should be deleted.
Traditionally,
the goal is formulated at the end of the introduction. L 58-61. Correct.
The fragment of L. 70-74 is appropriate in the conclusion, but not in the Introduction. Moreover
, this conclusion contradicts the results obtained.
The explanations of L. 128-134 are inappropriate and should be deleted. The authors should admit their methodological mistakes, at least for future research, rather than refer to the chromatographic column. Moreover, there is an obvious contradiction in this justification. At first, «HP-5 capillary column that was selected allows reliable quantification of major fatty acids», however, further: «the obtained fatty-acid profiles should be considered semi-quantitative».
Incorrect designation: “C18:1n9 Omega-9 , C18:2n6C Omega-6”. Must be:: 18:1n-9, 18:2n-6 или 18:1ω9, 18:2ω6
Author Response
Comment 1: In general, the arguments about the dependence of the composition of fatty acids on the conditions of cultivation and modulation of the biosynthetic pathways of L. 328-380 did not improve the manuscript, but only introduced unsubstantiated information.
Response 1: We thank the reviewer for this comment. We believe that the influence of cultivation conditions on fatty acid composition is an important contextual factor for interpreting the observed profiles. However, we agree that Figure 4 might contain broader considerations that were not sufficiently focused on the data obtained in this study; therefore, in the revised manuscript, the figure 4 has been removed. The relevant explanation has been retained in the text and supported with the appropriate citations.
Comment 2: “microalgae composition” L. 13 What kind of composition do you mean? Lipid or protein or elemental or pigment etc composition? Correct.
Response 2: We thank the reviewer for this clarification request. In this context, we specifically referred to lipid composition. The text has been corrected accordingly to avoid ambiguity (L 13).
Comment 3: What does it mean “lipid-related components”? L. 27. Correct.
Response 3: We thank the reviewer for this important remark. The sentence has been corrected (L. 27).
Comment 4: What is it “in most experimental groups”? Correct.
Response 4: We thank the reviewer for this comment. We have specified this sentence (L28-29).
Comment 5: do not recommend including Latin names of algae in keywords, as the lipid and fatty acid analysis of algae was carried out with serious errors.
Response 5: We thank the reviewer for this suggestion. The inclusion of the Latin names of the studied algae in the keywords was made following a previous recommendation during the review process to improve indexing accuracy and searchability of the manuscript. Therefore, the authors consider it appropriate to retain them in the keywords.
Comment 6: At least, the fragment of L. 40-52 is too far from the results of this study, so it should be deleted.
Response 6: We thank the reviewer for this observation. We agree that this fragment was not sufficiently aligned with the results of the present study. Therefore, the information related to human health has been removed to improve the focus and coherence of the manuscript.
Comment 7: Traditionally, the goal is formulated at the end of the introduction. L 58-61. Correct.
Response 7: We thank the reviewer for this comment. The aim of the study has now been repositioned to the end of the Introduction section in accordance with standard manuscript structure.
Comment 8: The fragment of L. 70-74 is appropriate in the conclusion, but not in the Introduction. Moreover, this conclusion contradicts the results obtained.
Response 8: We thank the reviewer for this comment. The indicated fragment (Lines 70–74) has been removed from the Introduction to improve the structure.
Comment 9: The explanations of L. 128-134 are inappropriate and should be deleted. The authors should admit their methodological mistakes, at least for future research, rather than refer to the chromatographic column. Moreover, there is an obvious contradiction in this justification. At first, «HP-5 capillary column that was selected allows reliable quantification of major fatty acids», however, further: «the obtained fatty-acid profiles should be considered semi-quantitative».
Response 9: We thank the reviewer for this careful and constructive comment. Our intention was to indicate that the HP-5 column is suitable for reliable quantification of the major fatty acids under standardized conditions, while at the same time recognizing the limitations of the method for comprehensive lipid characterization. To further support the validity of the used column, we are attaching the results of the FA standard analyses for the reviewer's consideration. However, to avoid ambiguity and overinterpretation, this section has been revised. The contradictory statements have been removed, and the methodological limitations are now clearly and consistently described.
Comment 10: Incorrect designation: “C18:1n9 Omega-9 , C18:2n6C Omega-6”. Must be:: 18:1n-9, 18:2n-6 или 18:1ω9, 18:2ω6
Response 10: We thank the reviewer for pointing out this incorrect designation. The fatty acid nomenclature has been corrected throughout the manuscript to the standard format (18:1n-9 and 18:2n-6).
Author Response File: Author Response.pdf
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for Authors
Manuscript ID: 4123949
Title: Microalgal Lipid Profile and Their Dietary Impact on Drosophila melanogaster.
Authors: Svitlana Miros, Svitlana Bilokon, Yiting Han and Ronald Halim.
Dear Authors,
Thank you for submitting a thoroughly revised version of your manuscript and for your detailed, point-by-point responses to my comments. I appreciate the care and attention you have given to addressing my concerns. In particular, it’s good that you have found and corrected the calculation error related to the dilution factor in the GC-FID quantification, which significantly improved the consistency between the gravimetric lipid data and the fatty acid sums. I acknowledge that you have provided clear justifications for the methodological choices, including the use of the HP-5 column. I accept your explanation that it serves as a screening tool for comparative profiling, and I appreciate the explicit addition of the method's limitations in the revised text, noting that it provides a semi-quantitative characterization. Your argument that the simplified FA pattern is a result of the specific, nutrient-replete cultivation conditions, rather than an analytical failure, is now well-supported by the literature and clearly explained in the revised manuscript. With these substantial improvements and clarifications, I believe the manuscript is now suitable for publication.
One minor suggestion: Figure 4 in the Discussion section does not appear to add essential information and could be removed.
As a general note for your future work, I would encourage you to consider these methodological aspects in advance to further strengthen your research.
I have no further comments on the work.
Author Response
Comment 1: One minor suggestion: Figure 4 in the Discussion section does not appear to add essential information and could be removed.
Response 1: We thank the reviewer for this suggestion. Following this recommendation, Figure 4 has been removed from the Discussion section. The relevant explanation has been retained in the text with the appropriate citation.
Reviewer 5 Report (New Reviewer)
Comments and Suggestions for Authors
I think the authors have responded to all raised questions and the manuscript has improved. I really appreciated the addition of a few experiments on the antioxidant properties of their microalgae extracts.
I want to add only a minor suggestion, Figure 4 seems to convey too much information, either make it more simpler and related to what was explained in the main text, or highlight in some way where the focus is (moreover, make it bigger because in the current size is difficult to read).
Author Response
I think the authors have responded to all raised questions and the manuscript has improved. I really appreciated the addition of a few experiments on the antioxidant properties of their microalgae extracts.
Comment 1: I want to add only a minor suggestion, Figure 4 seems to convey too much information, either make it more simpler and related to what was explained in the main text, or highlight in some way where the focus is (moreover, make it bigger because in the current size is difficult to read).
Response 1: We thank the reviewer for this helpful suggestion. To improve clarity and avoid overloading the manuscript with excessive schematic information, Figure 4 has been removed. The relevant process is now described concisely in the main text with the appropriate citation. We believe this revision improves readability and better aligns the content with the focus of the study.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This manuscript presents a timely and interesting study on the lipid profiles of four microalgae strains and their subsequent effects on the health parameters of Drosophila melanogaster. However, several key issues need to be addressed to strengthen the manuscript's clarity, methodological rigor, and interpretation of results before it can be considered for publication.
- A brief mention of more recent reviews or studies (post-2020) on microalgal fatty acids and their nutraceutical applications would strengthen the background.
-
The chosen concentration of 3 mg/mL (or 0.3% w/v) is reasonable, but without testing multiple concentrations, it is difficult to ascertain if this is the optimal dose or if higher/lower doses could have different (e.g., negative) effects. This should be acknowledged as a limitation in the discussion.
-
The volume of the 1L reactors is stated, but the working volume is not specified. The initial inoculation density (OD or cell count) is missing. The method for measuring light intensity (e.g., at the surface of the vessel?) should be clarified.
-
The method references a modified Bligh & Dyer, but the specific modifications are not detailed (e.g., solvent ratios, number of extraction steps). The number of biological replicates for lipid analysis is stated as n=2 in Table 1, which is very low. This should be increased to a minimum of n=3 for robust statistical analysis. The statement "Values are means ± SD (n=2)" in Table 1 is a major concern.
- melanogaster Assay: The sample size for the lifespan assay is good (n=100 per sex). However, for body mass and fecundity, it is only stated that n=5 vials were used. It is unclear how many individual flies were weighed per vial to get the average, or how many couples were used for the fecundity assay (n=5 pairs?). This must be explicitly stated.
-
Table 1 is cited as having n=2 replicates. The statistical groupings (A, B, C) based on a one-way ANOVA with n=2 are not statistically valid or reliable. This is a critical flaw. The authors must either repeat the lipid analysis with more biological replicates (n≥3) or remove the statistical annotations and rephrase the results to be purely descriptive.
-
The conclusion that "dietary supplementation with microalgae at 0.3%... had a positive effect" is slightly overstated. The effect was overwhelmingly positive for lifespan, neutral for body mass, and positive for only two of the four strains for fecundity. The negative effect of N. limnetica on female body mass should also be incorporated into the overall conclusion to provide a balanced view.
-
There are minor grammatical errors and awkward phrasings that should be corrected during a final proofread. For example:
Page 4: "Unsaturated FAs C15:0, C16:0 – others that were detected..." -> "Other saturated FAs detected in all strains included C15:0 and C16:0."
Page 6: "DNA was detected" -> This is likely a typo for "DHA".
Reviewer 2 Report
Comments and Suggestions for Authors
The submitted manuscript “Microalgal Lipid Profile and Their Dietary Impact on Drosophila melanogaster” by Svitlana Miros with coauthors is devoted to the analysis of total lipid content and fatty acids of four species of microalgae and the study of the effect of dietary supplementation of these microalgae on the health of D. melanogaster.
The main problem of the study is that the authors have poor knowledge in lipid biochemistry. The authors made serious methodological mistakes, which led to erroneous results.
There is extensive information about the lipids and fatty acids of microalgae, including the species studied in the submitted manuscript. It is well known that fatty acid composition of the algae depends on their taxonomic position. The specific features of algae classes are pronounced. For example, species belonging to the Nannochloropsis genus are typically rich in eicosapentaenoic acid (20:5n-3). Nannochloropsis limnetica (Eustigmatophyceae) also contains a high amounts of 20:5n-3 and another essential PUFA, arachidonic acid (20:4n-6). A distinctive feature of Chlorophyceae - green algae (Chlorella vulgaris, Scenedesmus communis and Tetradesmus obliquus) is an abundance of C18 PUFA, such as 18:2n-6, 18:3n-3, and18:4n-3, as well as the presence of C16 PUFA, n-6 and n-3 families. However, in the study, instead of the well-known PUFAs, the authors found a dominance of 15:0 characteristic of bacteria.
Regarding the effect of microalgae supplements on the health of D. melanogaster, no relationship was found between lipids and fatty acids of algae supplements and changes in the physiological parameters of drosophila. Therefore, the conclusion that “our study addresses an important gap by evaluating the nutritional value of microalgae using D.melanogaster” is unreasonable and too hasty.
In general, the manuscript does not provide new insights in this field.
My detailed comments:
The name of the species must be written in full at the first mention.
19-20. The authors do not know the classification of fatty acids. I recommend studying the classification of fatty acids by degree of unsaturation and making corrections to the manuscript. Here and further in the text and in the table.
Please indicate whether these algae are freshwater or marine.
A detailed description of the results is inappropriate in the Abstract, and there is no analysis of the data obtained and no conclusion.
Why did the authors name these algal species as oleaginous microalgal strains? According to the authors, can any organisms containing lipids be called oleaginous?
Traditionally, f2 medium is used for cultivation. What is 3 N-BBM +V medium? Why it was used in the study?
In addition, “Cultures were grown for 21 days”. What stage of culture growth does this time period correspond to? I think this is too long-term cultivation. Justify your chose, it is important. It is well-known that fatty acid composition of microalgae depends on their stage of grown. Moreover, what was cell count at harvesting as well as maximum specific growth rate and maximum biomass concentration?
It is necessary to describe in detail “a modified multi-step Bligh & Dyer procedure” for lipids extraction using the study.
“Transesterification was carried out with 2 M potassium methoxide and 20% HCl in methanol, followed by heating at 70 °C for 30 min” Did the authors use these reagents together? Describe in detail fatty acid methyl esters preparation, GLC analysis, identification and quantification. Please if the authors generally accepted or common methods, provide references with their description.
Discussion
169-173. It was in Introduction. Delete.
173. The authors should not repeat the detailed description of the results in the Discussion section.
176-190. The effect of cultivation conditions, medium etc. was not studied in the manuscript; therefore, it is inappropriate to discuss the effect of cultivation conditions on biomass and lipid levels.
190-202. This does not apply to the results obtained and therefore, it should be deleted.
203-208. The authors repeatedly return to the topic of the influence of culture conditions on the composition of lipids, although their research does not address this topic.
223-227. Redundancy. Similar to Introduction L. 37-42.
228-231. Redundancy. Similar to Introduction.
231-239. A detailed description of the results in the Discussion is inappropriate.
240-250 Previously, Qiu and colleagues demonstrated results, where feeding by 4 mg/mL wastewater-cultivated C. sorokiniana significantly extended the median lifespan of Drosophila [7]. In this regard, there is the contradiction with the conclusion: “The findings provide initial evidence that microalgae supplementation can positively in fluence key health parameters, underscoring their potential as a sustainable lipid source.”
247-250. “Thus, our study addresses an important gap by evaluating the nutritional value of microalgae using D.melanogaster, a model that remains largely underexplored in this context. “ I find this statement too ambitious and early.
To summarize, there is really no discussion of the results.
