Metal incorporated aminothiazole-derived compounds: synthesis, density function theory analysis, in vitro antibacterial and antioxidant evaluation

The present study advocates the combined experimental and computational study of metal-based aminothiazole-derived Schiff base ligands. The structure and electronic properties of ligands have been experimentally studied by spectroscopic methods (UV-Vis, FT-IR, 1H-NMR and 13C-NMR), mass spectrometry, elemental analysis and theoretically by density function theory (DFT). Computational calculations employing the B3LYP/6-31 + G(d,p) functional of DFT were executed to explore the optimized geometrical structures of ligands along with geometric parameters, molecular electrostatic potential (MEP) surfaces and frontier molecular orbital (FMO) energies. Global reactivity parameters estimated from FMO energy gaps signified the bioactive nature of ligands. The synthesized ligands were used for chelation with 3d-transition metals [VO(IV), Cr(III), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)] in 1 : 2 (metal : ligand) molar ratio. The spectral and magnetic results confirmed the formation of octahedral geometry around all the divalent and trivalent metal centres, whereas the tetravalent vanadyl centres were confirmed to have square-pyramidal geometry. All the as-synthesized compounds were investigated for in vitro antibacterial potential against two Gram-negative (Salmonella typhimurium and Escherichia coli) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria. Antibacterial assay results displayed pronounced activity, and their activity is comparable to that of a standard drug (streptomycin). The antioxidant potential of these compounds was assessed by employing diphenyl picryl hydrazide radical scavenging activity. The results displayed that all the metal chelates have exhibited more bioactivity in contrast with free ligands. The chelation was the main reason for their enhanced bioactivity. These results indicated that the thiazole metal-based compounds could be exploited as antioxidant and antimicrobial candidates.

complex. The stability of a complex (in DMF/PBS) can be qualitatively investigated by UV-visible spectroscopy by monitoring any spectral changes over a reasonable duration (say, up to 48 h). [7] The authors have presented the MS data only for the ligands. Why not for the complexes? The measurement of ESI-MS for the complexes and the observation of a prominent peak corresponding to the complex would also tell whether the complex is stable in a solution phase or not. [8] I have concerns regarding the stability of the VO(IV) complexes 1, 8, and 15. The two ligands are shown to bind the oxovanadium moiety as bidentate ligands. But, the ligand is forming a 3-membered ring. In coordination chemistry, 3-membered rings are generally strained and lead to the formation of unstable chelates unless stabilized by strong and special electronic factors. [9] Did the authors try to obtain the single-crystal X-ray diffraction structures of the complexes? It is a powerful and valuable tool in coordination chemistry. I strongly suggest that the authors should always try to report crystal structures (at least one or two if not all) in their future manuscripts. [10] NMR data of the ligands: please provide the coupling constant (J) values in the data section. [11] Please provide the DFT coordinates of the optimized structures in the supporting information. Besides, the HOMO/LUMOs of the remaining compounds should be provided as supporting information. [12] Antibacterial study: The zone-inhibition images for all the compounds should be provided as supporting information. [13] Figures 6 & 7: The error bars are really missing and should be added. [14] The graphical abstract looks are clumsy to me. The authors could present a simplified one by considering only three aspects, namely, DFT, antibacterial, and antioxidant studies. Similarly, the title of the manuscript could be improved. [15] I suggest that the authors go for one round of improvement of the English used in the manuscript in order to improve the readability Review form: Reviewer 2

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The synthesis of Schiff bases from the condensation of 2-amino-6-ethoxybenzothiazole or 2amino-6-nitrobenzothiazole with 5-chloroisatin or isatin and their 3d-transition metals complexes is reported in this ms. The compounds were characterized by spectroscopic methods (UV-Vis, FT-IR, 1H-NMR, 13CNMR), mass spectrometry, elemental analysis, and by computational calculations employing the B3LYP/6-31+G(d,p) functional of DFT. The in vitro antibacterial potential of the compounds is also reported.
The manuscript can be published in Royal Society Open Science only after major revision and reconsideration.
The crystal structures would be supportive for the characterization of the 24 compounds. Was any crystallization be attempted?
The structural and biological study of the Schiff base derived from 2-Amino-6methoxybenzothaizole with Isatin and its metal complexes has been already published by Hassan et.al. (2020 International Journal of Pharmaceutical Research 12(1), pp. 323-336). This work should be taken into consideration during the preparations of this manuscript. The Schiff base of 5-chloro isatin with 2-amino 5-nitrobenzothiazole and its complexes with ZnCl2 and CuCl2 are already reported by Shakir M et.al in Journal of Photochemistry and Photobiology B: Biology Volume 157, Pages 39 -56, 2016. Please rationalize the repetition of their quoting here. Similarly, the spectroscopic data have already reported.
Please check the electric charges balance of the formulae shown in Scheme 1. E.g Cr(III) needs 3 CH3COO-anions and not one as it is shown. There are two Cl-, counter anions in the case of Co(II). Similarly Co(II), Cu(II) and Ni(II) dichloride Please keep the chemical nomenclature. E.g In IR there are "vibrational" bands NMR resonance signals not "peaks", "peaks for the methylene", "carbon C4 peak" etc there are molecular fragments or ions in Mass spectrometry and not "peaks" etc Infra red and NMR Spectra A table with the characteristic vibrational bands and NMR signals of the compounds should be added in the text.

Molar conductance
Please check the consistence of the Molar conductance values (in table S2) and the formula of the compounds given in table S1. E.g. DMF solutions of electrolyte with 2 ions exhibit molar conductance value 65-90, while the corresponding value for the solution of electrolyte with 3 ions is 130-170 Ω-1cm2mol-1. The values measured correspond to 2 ions electrolydes which is not the case of proposed formulae in the case of Cr(III) acetate, copper dichloride, nickel dichloride cobalt dichloride etc.
Antibacterial activity Please give the diameter of the paper used in the inhibition zones (IZ). How the IZ diameter of e.g 4 mm in case of Z1 is measured? Figure 6. The negative values in the histograms of the antibacterial activity of aminothiazole ligands (Z1)-(Z3) vs their transition metal complexes (1)-(21) is not clear. Please explain.
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Dear Dr Sumrra: Title: Synthesis of Computed Aminothiazole Based Ligands and Their Endowed Transition Metal Chelates: In-vitro Antibacterial and Antioxidant Evaluation Manuscript ID: RSOS-210910 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: The reviewers have expressed some enthusiasm for this work, but have raised several valid concerns regarding the experimental details. In addition, Reviewer 2 has identified two existing publications that appear to detail similar results. Any revised manuscript should be prepared to avoid duplication of previously published results.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) In this paper, Sumrra and co-workers report on the development of a series of homoleptic Schiff base complexes of first-row transition metals, namely, VO(IV), Cr(III), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II). Theoretical DFT studies performed at a suitable level of theory have also been reported. The Schiff base ligands and the corresponding complexes were synthesized in satisfactory yields and characterized by routine methods. DFT studies were conducted to corroborate the experimental characterization data. The authors then went on to study the antibacterial efficacy of the compounds and demonstrated that the ligands and the metal chelates were significantly active against both gram (+) and gram (-) cultured strains. The activities were referenced to the standard antibiotic streptomycin. Similarly, several complexes were demonstrated to have antioxidant activity. The biological activities of the chelates were found to be higher compared to the ligands alone. Medicinal inorganic chemistry is a growing area of research and there is great scope the explore the potential of metal complexes for therapeutic applications. Similarly, antibacterial studies of new molecules with a novel mechanism of action are of importance considering the emergence of drug-resistant bacteria and AMR. After a careful review of the manuscript, I find that there are some important issues to address before I can recommend this work for publication. Addressing these issues will significantly improve the overall quality and the scientific soundness of the manuscript.
[1] My greatest concern is regarding the molar conductivity measurements in DMF. The conductivity values should be dependent on the electrolytic nature of the compounds and independent of the counteranion. The trend in conductivity should be 1:3 or 3:1 > 1:2 or 2:1 > 1:1. See for example, W. J. Geary, Coord. Chem. Rev., 7, 81 (1971). Thus, the trend observed for the complexes is not in line with the principle. Therefore, I suspect that the complexes might contain ionic impurities. The authors have not made any attempt to purify the complexes after synthesis.
[2] What is the likely mechanism of antibacterial and antioxidant activity of the complexes? What is the role of the central metal ion in the overall activity?
[3] The antibacterial activity should be quantified by determining the minimum inhibitory concentration (MIC) of the complexes. Besides, studies using cultured bacterial strains are now regarded as preliminary and do not provide a realistic assessment of the antibacterial potency. Bacterial biofilm formation is the main culprit for the development of antibiotic resistance and the severity of most infections that are difficult to treat (Nature Rev. Microbiol., 14, 563 (2016)). So, the compounds should be tested for their ability to inhibit bacterial biofilm formation on model surfaces.
[4] The antibacterial activity is normally due to the generation of ROS, while the antioxidant activity could be due to the lowing of ROS concentration by the compounds. Thus, a compound with a greater ability to generate ROS would more desirable as an antibacterial agent, while the one with greater ability to lower the concentration of ROS would be more desirable as an antioxidant. Thus, a compound showing both antibacterial and antioxidant activity seems to have a contradictory effect. The authors need to explain this fact.
[5] Why did the authors consider Cr(III) as the trivalent metal for their study? In my opinion, Fe(III) and Co(III) would have been more justified considering their bioessential nature. Cr(III) is not a good choice due to the fact that it can generate potentially carcinogenic other Cr species by speciation.
[6] Establishing the true identity and stability of a metal complex in a solution phase (preferably aqueous buffer such as PBS) is very important for any biological applications. The authors have not made any attempt to study the stability and solution identity of the reported complexes. Elemental analysis data do not provide any indication of the solution stability of a complex. The stability of a complex (in DMF/PBS) can be qualitatively investigated by UV-visible spectroscopy by monitoring any spectral changes over a reasonable duration (say, up to 48 h).
[7] The authors have presented the MS data only for the ligands. Why not for the complexes? The measurement of ESI-MS for the complexes and the observation of a prominent peak corresponding to the complex would also tell whether the complex is stable in a solution phase or not.
[8] I have concerns regarding the stability of the VO(IV) complexes 1, 8, and 15. The two ligands are shown to bind the oxovanadium moiety as bidentate ligands. But, the ligand is forming a 3membered ring. In coordination chemistry, 3-membered rings are generally strained and lead to the formation of unstable chelates unless stabilized by strong and special electronic factors.
[9] Did the authors try to obtain the single-crystal X-ray diffraction structures of the complexes? It is a powerful and valuable tool in coordination chemistry. I strongly suggest that the authors should always try to report crystal structures (at least one or two if not all) in their future manuscripts.
[10] NMR data of the ligands: please provide the coupling constant (J) values in the data section.
[11] Please provide the DFT coordinates of the optimized structures in the supporting information. Besides, the HOMO/LUMOs of the remaining compounds should be provided as supporting information.
[12] Antibacterial study: The zone-inhibition images for all the compounds should be provided as supporting information.
[13] Figures 6 & 7: The error bars are really missing and should be added. [14] The graphical abstract looks are clumsy to me. The authors could present a simplified one by considering only three aspects, namely, DFT, antibacterial, and antioxidant studies. Similarly, the title of the manuscript could be improved.
[15] I suggest that the authors go for one round of improvement of the English used in the manuscript in order to improve the readability Reviewer: 2 Comments to the Author(s) The synthesis of Schiff bases from the condensation of 2-amino-6-ethoxybenzothiazole or 2amino-6-nitrobenzothiazole with 5-chloroisatin or isatin and their 3d-transition metals complexes is reported in this ms. The compounds were characterized by spectroscopic methods (UV-Vis, FT-IR, 1H-NMR, 13CNMR), mass spectrometry, elemental analysis, and by computational calculations employing the B3LYP/6-31+G(d,p) functional of DFT. The in vitro antibacterial potential of the compounds is also reported.
The manuscript can be published in Royal Society Open Science only after major revision and reconsideration.
The crystal structures would be supportive for the characterization of the 24 compounds. Was any crystallization be attempted?
The structural and biological study of the Schiff base derived from 2-Amino-6methoxybenzothaizole with Isatin and its metal complexes has been already published by Hassan Please check the electric charges balance of the formulae shown in Scheme 1. E.g Cr(III) needs 3 CH3COO-anions and not one as it is shown. There are two Cl-, counter anions in the case of Co(II). Similarly Co(II), Cu(II) and Ni(II) dichloride Please keep the chemical nomenclature. E.g In IR there are "vibrational" bands NMR resonance signals not "peaks", "peaks for the methylene", "carbon C4 peak" etc there are molecular fragments or ions in Mass spectrometry and not "peaks" etc Infra red and NMR Spectra A table with the characteristic vibrational bands and NMR signals of the compounds should be added in the text.

Molar conductance
Please check the consistence of the Molar conductance values (in table S2) and the formula of the compounds given in table S1. E.g. DMF solutions of electrolyte with 2 ions exhibit molar conductance value 65-90, while the corresponding value for the solution of electrolyte with 3 ions is 130-170 Ω-1cm2mol-1. The values measured correspond to 2 ions electrolydes which is not the case of proposed formulae in the case of Cr(III) acetate, copper dichloride, nickel dichloride cobalt dichloride etc.
Antibacterial activity Please give the diameter of the paper used in the inhibition zones (IZ). How the IZ diameter of e.g 4 mm in case of Z1 is measured? Figure 6. We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Sumrra: Title: Metal Incorporated Aminothiazole Derived Compounds: Synthesis, DFT Analysis, In-vitro Antibacterial and Antioxidant Evaluation Manuscript ID: RSOS-210910.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. Authors' reply: We appreciate your kindness for conveying the reviewer comments on our manuscript and overall facilitation regarding the reviewing process of the manuscript. We have clarified (in reply of 2 nd comment) to Reviewer 2 and would also like to elaborate here that we have synthesized three aminothiazole Schiff base; (Z1) from 2-amino-6ethoxybenzothiazole and isatin, (Z2) from 2-amino-6-ethoxybenzothiazole and 5-chloroisatin, and (Z3) from 2-amino-6-nitrobenzothiazole and 5-chloroisatin. It can be clearly seen that we have synthesized new Schiff bases using aminothiazole moieties which are different from those used in the previously published recommended papers (2-amino-6methoxybenzothaizole and 2-amino-5-nitrobenzothiazole). Therefore, there is no duplication of previously published results.
The envisioned manuscript has been edited according to the comments/suggestions of the referees. The point-wise responses to the reviewers' comments are given in BLUE in 'Reply to reviewers' comments, while the revision in the revised manuscript has been highlighted as YELLOW. We hope that the reviewers' comments are satisfactorily addressed and we look forward to see our article published in Royal Society Open Science.

Reviewer(s)' Comments to Author:
Reviewer: 1 In this paper, Sumrra and co-workers report on the development of a series of homoleptic After a careful review of the manuscript, I find that there are some important issues to address before I can recommend this work for publication. Addressing these issues will significantly improve the overall quality and the scientific soundness of the manuscript.

Authors' reply:
We would like to acknowledge the reviewer for the encouraging review of our manuscript, and for the comments, corrections and suggestions that ensued. We also like to assure that all the concerns of the reviewer have been addressed in the best interest of the authors and readers. We have revised the text carefully in the light of the suggestions of the reviewer.
1. My greatest concern is regarding the molar conductivity measurements in DMF. The conductivity values should be dependent on the electrolytic nature of the compounds and independent of the counter anion. The trend in conductivity should be 1:3 or 3:1 > 1:2 or 2:1 > 1:1. See for example, W. J. Geary, Coord. Chem. Rev., 7, 81 (1971). Thus, the trend observed for the complexes is not in line with the principle. Therefore, I suspect that the complexes might contain ionic impurities. The authors have not made any attempt to purify the complexes after synthesis.

Authors' reply:
We appreciate the reviewer for this detailed clarification. We have reanalyzed the molar conductivity of complexes using the same solvent (DMF). Here we want to explain that previously we have compared the molar conductivity of complexes considering per ion in the outer sphere. But now in the revised version, we have discussed the molar conductivity of the whole complexes.

What is the likely mechanism of antibacterial and antioxidant activity of the complexes?
What is the role of the central metal ion in the overall activity?
Authors' reply: The most of the organic compounds that exhibit only one-or twodimensional topologies, whereas metal complexes have the potential that they can create three-dimensional structures using coordination chemistry of metal centers. Thus, metal We have also included this brief explanation about the mechanism of antibacterial and antioxidant activity of the complexes and the role of the central metal ion in the overall activity in the introduction.
3. The antibacterial activity should be quantified by determining the minimum inhibitory concentration (MIC) of the complexes. Besides, studies using cultured bacterial strains are now regarded as preliminary and do not provide a realistic assessment of the antibacterial potency. Bacterial biofilm formation is the main culprit for the development of antibiotic resistance and the severity of most infections that are difficult to treat (Nature Rev. Microbiol., 14, 563 (2016)). So, the compounds should be tested for their ability to inhibit bacterial biofilm formation on model surfaces.
Authors' reply: It would be interesting to explore this aspect. But because of the increasing number of COVID cases and a high mortality rate due to this severe 4 th layer, there is a strict lockdown here in Pakistan and here working in our university is almost online and there is no possibility to carry out this test at the moment. Therefore, we are unable to quantify the antibacterial activity by determining the minimum inhibitory concentration (MIC) of the complexes. 5. Why did the authors consider Cr(III) as the trivalent metal for their study? In my opinion, Fe(III) and Co(III) would have been more justified considering their bioessential nature.
Cr(III) is not a good choice due to the fact that it can generate potentially carcinogenic other Cr species by speciation.

Authors' reply:
We would like to clarify that as synthesis was carried out in inert atmosphere using anhydrous chemicals and divalent metal salts of iron and cobalt. As a result, the synthesized complexes are more justified with Fe(II) and Co(II) ions.
Furthermore, their colors, magnetic moments and electronic spectra confirmed their divalent nature. We appreciate the reviewer's insightful suggestion regarding the bioessential nature of Fe(III) and Co(III) ions and we will work on it in our next research work.
Cr(III) is a trace element in humans and plays a major role in glucose and fat metabolism.
The beneficial effects of Cr(III) in obesity and types 2 diabetes are known. It has been long considered an essential element, but now it has been reclassified as a nutritional supplement. On the other hand, Cr(VI) is a human carcinogen and exposure to it occurs both in occupational and environmental contexts. It induces also epigenetic effects on DNA, histone tails and microRNA; its toxicity seems to be related to its higher mobility in soil and swifter penetration through cell membranes than Cr (

Authors' reply:
We agree with the reviewer that 3-membered ring systems are generally more strained and least stable. But herein this manuscript, our VO(IV) complexes (1), (8), and (15) are 4-membered ring systems, which are quite stable and less strained than that of 3-membered ring systems. 9. Did the authors try to obtain the single-crystal X-ray diffraction structures of the complexes? It is a powerful and valuable tool in coordination chemistry. I strongly suggest that the authors should always try to report crystal structures (at least one or two if not all) in their future manuscripts.

Authors' reply:
We have tried our best to grow the single crystals of the respective metal complexes by changing different solvent ratios and other conditions. Unfortunately, we couldn't succeeded to get fine single crystal from the powder metal complexes. Then, all the ligands (Z1)-(Z3) and their selected metal complexes have been optimized by DFT to insight their geometries in absence of their well resolved SC-XRD data. Authors' reply: We agree with the reviewer and in response of this comment, we have included DFT coordinates of the optimized structures in the supporting information as Tables S7-S12. We have also summarized the optimized bond lengths and bond angles of studied compounds in Table S13. Moreover, the HOMO/LUMOs of all the other compounds have also been provided in the supporting information as Figures S23-S25. 12. Antibacterial study: The zone-inhibition images for all the compounds should be provided as supporting information.

Authors' reply:
We have added zone-inhibition images of representative compounds against each bacterial strain in the supporting information as Figure S28.
13. Figures 6 & 7: The error bars are really missing and should be added.

Authors' reply:
Thank you for pointing out this. We have revised both the suggested figures (6 & 7) by adding error bars and presented figures are now more accurate.

Reviewer: 2 Comments to the Author(s)
The synthesis of Schiff bases from the condensation of 2-amino-6-ethoxybenzothiazole or 2amino-6-nitrobenzothiazole with 5-chloroisatin or isatin and their 3d-transition metals complexes is reported in this ms. The compounds were characterized by spectroscopic methods (UV-Vis, FT-IR, 1H-NMR, 13CNMR), mass spectrometry, elemental analysis, and by computational calculations employing the B3LYP/6-31+G(d,p) functional of DFT. The in vitro antibacterial potential of the compounds is also reported. The manuscript can be published in Royal Society Open Science only after major revision and reconsideration.

Authors' reply:
We are grateful to the reviewer for the constructive comments about the paper. A major revision of the paper has been carried out to take all of them into account.
And in the process, we believe the paper has been significantly improved.
1. The crystal structures would be supportive for the characterization of the 24 compounds.

Was any crystallization be attempted?
Authors' reply: Initially, the precipitates were recrystallized using equimolar ratio of ethanol and ether to get pure product. After that to get the crystals, we have tried some more solvent mixtures varying their ratios, but we couldn't succeeded to get fine single crystal. Then, all the ligands (Z1)-(Z3) and their selected metal complexes have been optimized by DFT to insight their geometries in absence of their well resolved SC-XRD data.
2. The structural and biological study of the Schiff base derived from 2-Amino-6methoxybenzothaizole with Isatin and its metal complexes has been already published by Authors' reply: Thank you for this detailed explanation. We have again performed the molar conductance measurement for complexes using the same solvent (DMF). And now we have compared the molar conductance values of complexes considering all the electrolytes. But in the previous version, we have discussed the molar conductivity of complexes considering per ion in the outer sphere.

Antibacterial activity
Please give the diameter of the paper used in the inhibition zones (IZ). How the IZ diameter of e.g 4 mm in case of Z1 is measured?
Authors' reply: The reviewer has raised an important point here. The diameter of the paper used in the inhibition zones (IZ) is 6 mm. As we have mentioned in the experimental detail of antibacterial activity that "At the end, clear or inhibition zones were noted (in millimeter) for all the tested compounds and standard drug against each bacterial strain." We have not compared the overall diameter of the inhibition zones. We have only discussed the clear zone which is definitely the actual area representing the inhibitory activity of compounds.
In the case of (Z1), the diameter of inhibition zone was 10 mm, but the clear zone area was 4 mm (after subtracting the diameter of paper as shown in Figure S28). 8. Figure 6. The negative values in the histograms of the antibacterial activity of aminothiazole ligands (Z1)-(Z3) vs their transition metal complexes (1)-(21) is not clear.

Please explain.
Authors' reply: The negative values for diameter of inhibition zone in Figure 6 represent the antibacterial activity of all the compounds against Gram-negative bacterial strains (Escherichia coli and Salmonella typhimurium). Actually, the inhibition values were not negative, but these are graphically represented as negative for Gram-negative bacteria in Figure 6.
At the end, we would like to thank both the reviewers again for taking the time to review our manuscript and helping us to improve the consistency of our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. In addition to the above comments, all spelling and grammatical errors have been corrected throughout the manuscript. And overall, the quality of the manuscript has been enhanced. We hope that the manuscript would now be suitable for acceptance and publication in Royal Society Open Science. I look forward hearing your positive response.