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Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research


Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at − 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species.

Key points

• First report on effects of preservation methods on fish larval microbiome profiles.

• First report on activity ranking of preservation methods based on metagenomics metrics.

• Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

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Data availability

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.

Code availability

All the metagenomic data sets are available on the NCBI Sequence Read Archive under the SRA study SRP338188 (Accession numbers SRR16005287-SRR16005298).


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The authors are grateful to the Head, Marine Biotechnology, Fish Nutrition and Health Division, the Director, ICAR-CMFRI, Kochi, and the Head-in-Charge of Mandapam Regional Centre of ICAR-CMFRI for providing the research facilities. GS and ING acknowledge the DBT-funded project entitled “Dr. E.G. Silas Centre of Excellence and Innovation (Dr. EGS-CoEI) in Marine Fish Microbiome and Nutrigenomics” for the fellowship awarded to them.


This work was supported by the Department of Biotechnology, Government of India funded project “Dr. E.G. Silas Centre of Excellence and Innovation (Dr. EGS-CoEI) in Marine Fish Microbiome and Nutrigenomics” (BT/AAQ/3/SP28267/2018).

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Authors and Affiliations



STG and SRK conceptualized the presented idea, supervised the findings, analyzed the results, and wrote the manuscript. GS performed the bioinformatics analysis of the data. SE and RKJ helped in the sampling and sample processing. AKK, ARNP, RKP, SM, and TG performed cobia larval production and maintenance. AG, SRK, and PVG supervised the project and acquired financial support for the project leading to this publication.

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Correspondence to S. R. Krupesha Sharma.

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Ethics approval

The live larvae for the experiments were handled in consonance with the guidelines of the UK Animals (Scientific Procedures) Act (1986) and the EU Directive 2010/63/EU for animal experiments (2019). The experimental protocols for the maintenance of fish larvae were approved by the ICAR-CMFRI, Kochi, India (BT/AAQ/3/SP28267/2018).

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Sumithra, T.G., Sharma, S.R.K., Gayathri, S. et al. Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research. Appl Microbiol Biotechnol 106, 4719–4735 (2022).

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  • Cobia
  • Larvae
  • Metagenomics
  • Preservation
  • Absolute ethanol