A Lipidomic Profile of a Sustainable Source of Omega-3 Long-Chain Polyunsaturated Fatty Acids, Greenshell Mussels™, Perna canaliculus
Round 1
Reviewer 1 Report
This manuscript entitled "A Lipidomic Exploration of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™,Perna canaliculus" by Taylor et al. is an interesting manuscript focused on use new analytical technique to obtain lipid classes profile on commercial bivalve
In general, the manuscript is well written. Introduction provides the justification, material and methods seems to be clear and complete, results are well represented and discussion as well to conclusion are clear. It is also clear that the results show the new analytical techniques with great potential for the valuable information on the different lipid classes obtained.
However, some minimal details could be attained to improve the manuscript.
The paper is focused on evaluating new analytical techniques, which is stipulated in the objective of the work. However, the experimental design generates an in-depth analysis of the lipid profile differences between tissues of the model bivalve. That said, the title could be modified, because even the discussion focuses more on the differences between tissues and not so much on the differences in the analytical power of the new tools. As a simple proposal or idea: Lipidomic Profile of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™,Perna canaliculus.at least, the objective should include the comparison between tissues that is made in the work.
Line 164.- ripeness and plumpness. Are there any values or registers that allow authors to report indices according to these parameters?
Table 1 does not make clear what is the difference between the two columns (what is the comparative?). In the same table, where there is no data for moisture and lipids, it is recommended to complete the table and fill in those spaces with an ND (not determined). It is also recommended to make it clear that the analyses shown are on a wet basis.
In the figure caption 2. The x-axis may initially be confusing. It is recommended to include a brief explanation of the x-axis key arrangement. Where I assume the three fatty acids that make up the triglyceride are denoted. You could add something like: Type (sn-1_sn-2_sn3) where sn means….. The same adjustment for all the other figure captions!!
Line 287: sn-3. Sn-1, sn-2 or sn-3 is not defined.
Author Response
Thanks for the review
We have reviewed the recommendations and have addressed all of them as suggested by the reviewers. These have all been made using track changes on the document downloaded from the MDPI site as directed. As directed, we are providing a brief summary of the changes that we have been made and the editors and reviewers can check these against the manuscript with track changes.
This manuscript entitled "A Lipidomic Exploration of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™,Perna canaliculus" by Taylor et al. is an interesting manuscript focused on use new analytical technique to obtain lipid classes profile on commercial bivalve
In general, the manuscript is well written. Introduction provides the justification, material and methods seems to be clear and complete, results are well represented and discussion as well to conclusion are clear. It is also clear that the results show the new analytical techniques with great potential for the valuable information on the different lipid classes obtained.
However, some minimal details could be attained to improve the manuscript.
The paper is focused on evaluating new analytical techniques, which is stipulated in the objective of the work. However, the experimental design generates an in-depth analysis of the lipid profile differences between tissues of the model bivalve. That said, the title could be modified, because even the discussion focuses more on the differences between tissues and not so much on the differences in the analytical power of the new tools. As a simple proposal or idea: Lipidomic Profile of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™,Perna canaliculus.at least, the objective should include the comparison between tissues that is made in the work.
Corrected title and altered aim sentence to read “This study adds to the traditional lipid analytical outputs with new lipidomic capabilities to interrogate the lipid species differences between tissues rich in oil.”
Line 164.- ripeness and plumpness. Are there any values or registers that allow authors to report indices according to these parameters?
This measurement is objective but in this case was done by Rodney Roberts who has 20+ years’ experience in the industry and based on the work previously done by Buchanan (2001) Measuring reproductive condition in the Greenshell (TM) mussel Perna canaliculus S. Buchanan New Zealand Journal of Marine and Freshwater Research 2001 Vol. 35 Issue 5 Pages 859-870
Table 1 does not make clear what is the difference between the two columns (what is the comparative?). In the same table, where there is no data for moisture and lipids, it is recommended to complete the table and fill in those spaces with an ND (not determined). It is also recommended to make it clear that the analyses shown are on a wet basis.
This table is comparing the three organs, the left hand column is looking as a proportion of the whole GSM while the right hand column is looking at the proportion of the extracted oil. Changes to the table has also been made to clarify.
In the figure caption 2. The x-axis may initially be confusing. It is recommended to include a brief explanation of the x-axis key arrangement. Where I assume the three fatty acids that make up the triglyceride are denoted. You could add something like: Type (sn-1_sn-2_sn3) where sn means….. The same adjustment for all the other figure captions!!
Line 287: sn-3. Sn-1, sn-2 or sn-3 is not defined.
Sentence added to the methods at line 164
The MS/MS data provide sufficient information to determine the acyl chain composition of each lipid species but not the stereospecific numbering (sn) of the acyl chain position on the 3 carbon glycerol backbone and thus denoted as “_” between acyl chains (“/” used when sn position is known).
Reviewer 2 Report
Comments:
Manuscript Sustainability-2354880 is an interesting work providing information about “Lipidomic Exploration of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™, Perna canaliculus”. The comment is listed as follows:
Materials and methods
Ln 155- What R software package and version was used in this study? State it!
Author Response
Thanks for the review
We have reviewed the recommendations and have addressed all of them as suggested by the reviewers. These have all been made using track changes on the document downloaded from the MDPI site as directed. As directed, we are providing a brief summary of the changes that we have been made and the editors and reviewers can check these against the manuscript with track changes.
Ln 155- What R software package and version was used in this study? State it!
New words in the MM to cover this
Further analysis was conducted using in-house R scripts (R version 4.2.1, using tidyverse package [23,24]) for data normalization of Compound Discoverer (ThermoFisher) results and to aid in the identification of lipid species. Probabilistic quotient normalisation (PQN) was applied to normalise the data without the potential introduction of artificial correlations in the data [25]. Missing values were removed from the data analysis. Statistical analysis and plotting was conducted using the R packages stats, emmeans, ggplot2, cowplot, GGally, ggfortify and RColorBrewer packages. The significance between tissues was determined for compounds that were present in more than half of the samples and were present in all three tissues using ANOVA and Tukey HSD of the log-transformed data. Principal component analysis (PCA) was conducted singular value decomposition using the “prcomp” function in the R Stats package. Correlation analysis was conducted using the “pairs” function in R graphics package and the Pearson correlation coefficient calculated using the “cor” function in the R Stats package.
Reviewer 3 Report
Review of the paper "A Lipidomic Exploration of a Sustainable Source of Omega-3 Long-chain Polyunsaturated Fatty Acids, Greenshell Mussels™, Perna canaliculus" by Matthew C. Taylor, Rodney Roberts, and Matthew R. Miller submitted to "Sustainability".
General comment.
The species Perna canaliculus (green shell mussel) is a member of the class Bivalvia in the phylum Mollusca within the family Mytilidae. In New Zealand, green shell mussels are farmed using a longline technique. The New Zealand Greenshell mussel industry currently produces ~33,000 tonnes weight per year and is valued at over NZ$305 million. The majority of the harvested mussels are exported as frozen or fresh food, although the reported bioactivity in the oil of Perna canaliculus has increased industry interest in this species for pharmaceutically useful compounds. A number of bioactive compounds have been found in the extract of Perna canaliculus species, which is emerging as the most carefully and intensively studied compound for medicinal and therapeutic effects. Perna canaliculus has been used in the development of commercial therapeutic agents for arthralgia in both humans and animals. There are also several minor lipid components in GSM oil, including non-methylene-interrupted (NMI) FA, plasmalogen, phytosterols and furan fatty acids. These minor lipids are not present in most fish oil products and some have been shown to have beneficial effects on human health. However, little is known about other minor lipid components, and to fill this gap, the authors conducted a laboratory study to determine the content and activity of the lipidome of green shell mussels using ultra-high performance liquid chromatography. The authors found 16 different lipid species in Perna canaliculus, including ceramide aminoethyl phosphonate. Many lipid species were shown to be differentially expressed between tissues. They were able to identify lipid species that were associated with the digestive gland, the mantle or the gonad. The authors' conclusion was that this new data could be useful for aquaculture. The authors used standard and appropriate methods to treat the data. The main results are illustrated with relevant figures and tables. The discussion is comprehensive and focused on the main findings.
Abstract
L 10. Consider replacing “Greenshell mussels” with “Greenshell mussel”
L 11. Consider replacing “lipid extract” with “lipid extracts”
Introduction
L 37. Consider replacing “methods to product” with “methods to produce”
L 60. Consider replacing “PUFA and possible” with “PUFA and possibly”
The authors have provided a revenue value and a price for GSM oil in NZ$. They should update these values with equivalent values in US$.
Material and methods.
L 145. Consider replacing “Orbitrap fusion data was” with “Orbitrap fusion data were”
L 161. Consider replacing “log transformed data” with “log-transformed data”
The authors used Principal Component Analysis to reveal the data separation. The authors should include a reference to this method in the M&M. They should also indicate on which metric their analysis was based: Euclidean distance, Bray-Curtis or other.
The same is relevant to correlation analysis. It was used by the authors to show the relationship between the DG and TG pools, but no details are given in the M&M. What kind of correlation was used? Did the authors adjust p values for multiple correlations?
The authors should describe in detail any statistical methods used.
Results
The authors stated that the mussels were weighed and measured for length and width at the widest point (L 88), but no size data were given in the text. The authors should report not only averages but also the range for each parameter.
L 187. Consider replacing “normalised data was” with “normalised data were”
Section 3.2. The authors stated that they used principal component analysis to determine whether lipid composition was significantly different between the three tissue types (L 188). However, this method does not provide a p-value to assess whether a difference is significant. This method is used to visualize the data. This concern should be addressed in a revised version of the text.
L 208. Consider replacing “are most likely” with “is most likely”
L 220. Consider replacing “are the triglyceride” with “is the triglyceride”
L 227. Consider replacing “few significant difference” with “few significant differences”
L 250. Consider replacing “a relatively lower distribution” with “a relatively low distribution”
Discussion.
L 380. Consider replacing “The ecosystems” with “The ecosystem”
L 407. Consider replacing “lipids directly form” with “lipids directly from”
L 413. Consider replacing “is supported by supported by” is supported by”
L 415. Consider replacing “seasonal or locational” with “seasonal or spatial”
L 447. Consider replacing “is in accordance” with “are in accordance”
L 472. Consider replacing “reported for its” with “reported for their”
L 496. Consider replacing “All bivalves and including GSM” with “All bivalves including GSM”
L 503. Consider replacing “Plasmalogen PE species with EPA has” with “Plasmalogen PE species with EPA have”
L 516. Consider replacing “CAEP in blue mussel” with “CAEP in blue mussels”
Conclusion
This section should be shortened to focus on the key findings.
References.
The Latin names should be written in italics.
The paper is well-written.
Author Response
General comment.
The species Perna canaliculus (green shell mussel) is a member of the class Bivalvia in the phylum Mollusca within the family Mytilidae. In New Zealand, green shell mussels are farmed using a longline technique. The New Zealand Greenshell mussel industry currently produces ~33,000 tonnes weight per year and is valued at over NZ$305 million. The majority of the harvested mussels are exported as frozen or fresh food, although the reported bioactivity in the oil of Perna canaliculus has increased industry interest in this species for pharmaceutically useful compounds. A number of bioactive compounds have been found in the extract of Perna canaliculus species, which is emerging as the most carefully and intensively studied compound for medicinal and therapeutic effects. Perna canaliculus has been used in the development of commercial therapeutic agents for arthralgia in both humans and animals. There are also several minor lipid components in GSM oil, including non-methylene-interrupted (NMI) FA, plasmalogen, phytosterols and furan fatty acids. These minor lipids are not present in most fish oil products and some have been shown to have beneficial effects on human health. However, little is known about other minor lipid components, and to fill this gap, the authors conducted a laboratory study to determine the content and activity of the lipidome of green shell mussels using ultra-high performance liquid chromatography. The authors found 16 different lipid species in Perna canaliculus, including ceramide aminoethyl phosphonate. Many lipid species were shown to be differentially expressed between tissues. They were able to identify lipid species that were associated with the digestive gland, the mantle or the gonad. The authors' conclusion was that this new data could be useful for aquaculture. The authors used standard and appropriate methods to treat the data. The main results are illustrated with relevant figures and tables. The discussion is comprehensive and focused on the main findings.
Abstract
L 10. Consider replacing “Greenshell mussels” with “Greenshell mussel”
corrected
L 11. Consider replacing “lipid extract” with “lipid extracts”
corrected
Introduction
L 37. Consider replacing “methods to product” with “methods to produce”
corrected
L 60. Consider replacing “PUFA and possible” with “PUFA and possibly”
corrected
The authors have provided a revenue value and a price for GSM oil in NZ$. They should update these values with equivalent values in US$.
Corrected using conversion of 0.63 which is the 2023 average exchange rate
Material and methods.
L 145. Consider replacing “Orbitrap fusion data was” with “Orbitrap fusion data were”
corrected
L 161. Consider replacing “log transformed data” with “log-transformed data”
corrected
The authors used Principal Component Analysis to reveal the data separation. The authors should include a reference to this method in the M&M. They should also indicate on which metric their analysis was based: Euclidean distance, Bray-Curtis or other.
The same is relevant to correlation analysis. It was used by the authors to show the relationship between the DG and TG pools, but no details are given in the M&M. What kind of correlation was used? Did the authors adjust p values for multiple correlations?
The authors should describe in detail any statistical methods used.
Corrected - Reference included with the text below
Principal component analysis (PCA) was conducted singular value decomposition using the “prcomp”function in the R Stats package (R Core Team, 2022). Correlation analysis was conducted using the pairs function in R graphics package and the pearson correlation coefficient calculated using the cor function in the R Stats package. “
Results
The authors stated that the mussels were weighed and measured for length and width at the widest point (L 88), but no size data were given in the text. The authors should report not only averages but also the range for each parameter.
In the results the following data was added “The mussels selected for analysis were large females (average shell length 113 ± 11 mm, width 51± 5 mm) with marketable plumpness and colour (gonad visual grading score 6.6 ± 0.4).”
L 187. Consider replacing “normalised data was” with “normalised data were”
corrected
Section 3.2. The authors stated that they used principal component analysis to determine whether lipid composition was significantly different between the three tissue types (L 188). However, this method does not provide a p-value to assess whether a difference is significant. This method is used to visualize the data. This concern should be addressed in a revised version of the text.
“significantly” removed.
L 208. Consider replacing “are most likely” with “is most likely”
corrected
L 220. Consider replacing “are the triglyceride” with “is the triglyceride”
corrected
L 227. Consider replacing “few significant difference” with “few significant differences”
corrected
L 250. Consider replacing “a relatively lower distribution” with “a relatively low distribution”
corrected
Discussion.
L 380. Consider replacing “The ecosystems” with “The ecosystem”
corrected
L 407. Consider replacing “lipids directly form” with “lipids directly from”
corrected
L 413. Consider replacing “is supported by supported by” is supported by”
corrected
L 415. Consider replacing “seasonal or locational” with “seasonal or spatial”
corrected
L 447. Consider replacing “is in accordance” with “are in accordance”
corrected
L 472. Consider replacing “reported for its” with “reported for their”
corrected
L 496. Consider replacing “All bivalves and including GSM” with “All bivalves including GSM”
corrected
L 503. Consider replacing “Plasmalogen PE species with EPA has” with “Plasmalogen PE species with EPA have”
corrected
L 516. Consider replacing “CAEP in blue mussel” with “CAEP in blue mussels”
corrected
Conclusion
This section should be shortened to focus on the key findings.
Corrected
This paper focused on a qualitative analysis of the different GSM tissues to provide new results on the distribution of a range of lipid classes in this unique and commercially harvested species, and to demonstrate the utility of lipidomics alongside traditional lipid profile in such research and development. The traditional lipid profile is a concise data set and easily comparable to published results in lipid or broad nutritional studies. Conversely, lipidomic analysis in marine organisms is still in its infancy. Many standards are available but are more suited for the fatty acid composition of mammalian and plant-based lipids. Other lipids such as the CAEP have no available standards and are not present in lipidomic reference datasets, creating further challenges for studying lipidomics in marine systems. The lipidomic data set highlights the complexity of the lipidome and allows more detailed examination of the roles of specific lipids in metabolism and function. This could assist studies aimed at understanding the molecules and physiological processes underpinning the bioactivity of GSM powders and oils (10), and could ultimately help breeding programmes to produce GSM strains with enhanced bioactivity.
References.
The Latin names should be written in italics.
Corrected
