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

Applied Microbiology and Biotechnology volume 106, pages 4719–4735 (2022)Cite this article

Abstract

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).

References

  • Bakke I, Coward E, Andersen T, Vadstein O (2015) Selection in the host structures the microbiota associated with developing cod larvae (Gadus morhua). Environ Microbiol 17:3914–3924

    Article  Google Scholar 

  • Bjornsdottir R, Johannsdottir J, Coe J, Smaradottir H, Agustsson T, Sigurgisladottir S, Gudmundsdottir BK (2009) Survival and quality of halibut larvae (Hippoglossus hippoglossus L.) in intensive farming: possible impact of the intestinal bacterial community. Aquaculture 286:53–63

    Article  Google Scholar 

  • Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Bin KK, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu Y-X, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson CHD, Willis AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q, Knight R, Caporaso JG (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852–857

    Article  CAS  Google Scholar 

  • Bruggeling CE, Garza DR, Achouiti S, Mes W, Dutilh BE, Boleij A (2021) Optimized bacterial DNA isolation method for microbiome analysis of human tissues. Microbiologyopen 10:3

    Article  Google Scholar 

  • Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carroll IM, Ringel-Kulka T, Siddle JP, Ringel Y (2012) Alterations in composition and diversity of the intestinal microbiota in patients with diarrhea-predominant irritable bowel syndrome. NeurogastroenterolMotil 24:521-e248

    Article  CAS  Google Scholar 

  • Choo JM, Leong LE, Rogers GB (2015) Sample storage conditions significantly influence faecal microbiome profiles. Sci Rep 5:16350

    Article  CAS  Google Scholar 

  • De Cock M, Virgilio M, Vandamme P, Augustinos A, Bourtzis K, Willems A, De Meyer M (2019) Impact of sample preservation and manipulation on insect gut microbiome profiling. a test case with fruit flies (Diptera, Tephritidae). Front Microbiol 10:2833

  • Dominianni C, Wu J, Hayes RB, Ahn J (2014) Comparison of methods for fecal microbiome biospecimen collection. BMC Microbiol 14:103

    Article  Google Scholar 

  • Douglas GM, Maffei VJ, Zaneveld JR, Yurgel SN, Brown JR, Taylor CM, Huttenhower C, Langille MGI (2020) PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 38:685–688

    Article  CAS  Google Scholar 

  • Estrada UR, Yasumaru FA, Tacon AGJ, Lemos D (2016) Cobia (Rachycentron canadum): a selected annotated bibliography on aquaculture, general biology and fisheries 1967–2015. Rev Fish SciAquac 24:1–97

    Google Scholar 

  • FAO (2018) The state of world fisheries and aquaculture: meeting the sustainable developmental goals

  • Giatsis C, Sipkema D, Smidt H, Heilig H, Benvenuti G, Verreth J, Verdegem M (2015) The impact of rearing environment on the development of gut microbiota in tilapia larvae. Sci Rep 5:18206

    Article  CAS  Google Scholar 

  • Gopakumar G, Abdul Nazar AK, Tamilmani G, Sakthivel M, Kalidas C, Ramamoorthy N, Palanichamy S, Ashok Maharshi V, Srinivasa Rao K, Syda Rao G (2011) Broodstock development and controlled breeding of cobia Rachycentron canadum (Linnaeus 1766) from Indian seas. Indian J Fish 58:27–32

    Google Scholar 

  • Griffiths S, Melville K, Cook M, Vincent S, Pierre M, Lanteigne C (2001) Profiling of bacterial species associated with haddock larviculture by PCR amplification of 16S rDNA and denaturing gradient gel electrophoresis. J AquatAnim Health 13:355–363

    Google Scholar 

  • Hale VL, Tan CL, Knight R, Amato KR (2015) Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8weeks. J Microbiol Methods 113:16–26

    Article  Google Scholar 

  • Hammer DAT, Ryan PD, Hammer Ø, Harper DAT (2001) Past: paleontological statistics software package for education and data analysis. Palaeontol Electron 4(1):1–9

    Google Scholar 

  • Hickl O, Heintz-Buschart A, Trautwein-Schult A, Hercog R, Bork P, Wilmes P, Becher D (2019) Sample preservation and storage significantly impact taxonomic and functional profiles in metaproteomics studies of the human gut microbiome. Microorganisms 7:367

    Article  CAS  Google Scholar 

  • Ingala MR, Simmons NB, Wultsch C, Krampis K, Speer KA, Perkins SL (2018) Comparing microbiome sampling methods in a wild mammal: fecal and intestinal samples record different signals of host ecology, evolution. Front Microbiol 9:803

    Article  Google Scholar 

  • Ingerslev H-C, von GersdorffJørgensen L, Lenz Strube M, Larsen N, Dalsgaard I, Boye M, Madsen L (2014) The development of the gut microbiota in rainbow trout (Oncorhynchus mykiss) is affected by first feeding and diet type. Aquaculture 424–425:24–34

    Article  Google Scholar 

  • Jensen S, Øvreås L, Bergh Ø, Torsvik V (2004) Phylogenetic analysis of bacterial communities associated with larvae of the Atlantic Halibut propose succession from a uniform normal flora. Syst Appl Microbiol 27:728–736

    Article  CAS  Google Scholar 

  • LópezNadal A, Ikeda-Ohtsubo W, Sipkema D, Peggs D, McGurk C, Forlenza M, Wiegertjes GF, Brugman S (2020) Feed, microbiota, and gut immunity: using the zebrafish model to understand fish health. Front Immunol 11:114

    Article  Google Scholar 

  • Mackenzie BW, Waite DW, Taylor MW (2015) Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences. Front Microbiol 6:130

    Google Scholar 

  • Malla MA, Dubey A, Kumar A, Yadav S, Hashem A, Abd_Allah EF (2019) Exploring the human microbiome: the potential future role of next-generation sequencing in disease diagnosis and treatment. Front Immunol 9:2868.

  • Mehinto AC, Martyniuk CJ, Spade DJ, Denslow ND (2012) Applications for next-generation sequencing in fish ecotoxicogenomics. Front Genet 3:62

    Article  Google Scholar 

  • Nazar AKA, Jayakumar R, Tamilmani G, Sakthivel M, Ramesh Kumar, P Anikuttan KK, Sankar M (2019) Practical handbook on seed production of cobia and silver pompano. Manual. ICAR - Central Marine Fisheries Research Institute, Kochi.

  • Prakash S, Rodes L, Coussa-Charley M, Tomaro-Duchesneau C, Tomaro-Duchesneau C, Coussa-Charley, Rodes (2011) Gut microbiota: next frontier in understanding human health and development of biotherapeutics. Biol Targets Ther 71.

  • Rasheeda MK, Rangamaran VR, Srinivasan S, Ramaiah SK, Gunasekaran R, Jaypal S, Gopal D, Ramalingam K (2017) Comparative profiling of microbial community of three economically important fishes reared in sea cages under tropical offshore environment. Mar Genomics 34:57–65

    Article  CAS  Google Scholar 

  • Schultz CL, Akker Y, Du J, Ratech H (1999) A lysis, storage, and transportation buffer for long-term, room-temperature preservation of human clinical lymphoid tissue samples yielding high molecular weight genomic DNA suitable for molecular diagnosis. Am J Clin Pathol 111:748–752

    Article  CAS  Google Scholar 

  • Sinha R, Abu-Ali G, Vogtmann E, Fodor AA, Ren B, Amir A, Schwager E, Crabtree J, Ma S, Abnet CC, Knight R, White O, Huttenhower C (2017) Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium. Nat Biotechnol 35:1077–1086

    Article  CAS  Google Scholar 

  • Skjermo J, Bakke I, Dahle SW, Vadstein O (2015) Probiotic strains introduced through live feed and rearing water have low colonizing success in developing Atlantic cod larvae. Aquaculture 438:17–23

    Article  Google Scholar 

  • Song SJ, Amir A, Metcalf JL, Amato KR, Xu ZZ, Humphrey G, Knight R (2016) Preservation methods differ in fecal microbiome stability, affecting suitability for field studies. mSystems 1:3.

  • Sun Y-Z, Yang H-L, Ling Z-C, Ye J-D (2013) Microbial communities associated with early stages of intensively reared orange-spotted grouper (Epinephelus coioides). Aquac Res 46:131–140

    Article  Google Scholar 

  • Takasaki H, Maruyama K, Takahashi F, Fujita M, Yoshida T, Nakashima K, Myouga F, Toyooka K, Yamaguchi-Shinozaki K, Shinozaki K (2015) SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence. Plant J 84(6):1114–1123

    Article  CAS  Google Scholar 

  • The Human Microbiome Project Consortium (2012) Structure, function and diversity of the healthy human microbiome. Nature 486:207–214

    Article  Google Scholar 

  • Vadstein O, Attramadal KJK, Bakke I, Forberg T, Olsen Y, Verdegem M, Giatsis C, Skjermo J, Aasen IM, Gatesoupe F-J, Dierckens K, Sorgeloos P, Bossier P (2018) Managing the microbial community of marine fish larvae: a holistic perspective for larviculture. Front Microbiol 9:1820

    Article  Google Scholar 

  • Vadstein O, Bergh Ø, Gatesoupe FJ, Galindo-Villegas J, Mulero V, Picchietti S, Scapigliati G, Makridis P, Olsen Y, Dierckens K, Defoirdt T, Boon N, De Schryver P, Bossier P (2013) Microbiology and immunology of fish larvae. Rev Aquac 5:S1–S25

    Article  Google Scholar 

  • Vlčková K, Mrázek J, Kopečný J, Petrželková KJ (2012) Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla). J Microbiol Methods 91:45–51

    Article  Google Scholar 

  • Vogtmann E, Chen J, Kibriya MG, Chen Y, Islam T, Eunes M, Ahmed A, Naher J, Rahman A, Amir A, Shi J, Abnet CC, Nelson H, Knight R, Chia N, Ahsan H, Sinha R (2017) Comparison of fecal collection methods for microbiota studies in Bangladesh. Appl Environ Microbiol 83:10

    Article  Google Scholar 

  • Wanka KM, Damerau T, Costas B, Krueger A, Schulz C, Wuertz S (2018) Isolation and characterization of native probiotics for fish farming. BMC Microbiol 18:119

    Article  CAS  Google Scholar 

  • Wu C, Chen T, Xu W, Zhang T, Pei Y, Yang Y, Zhang F, Guo H, Wang Q, Wang L, Zhao B (2021) The maintenance of microbial community in human fecal samples by a cost effective preservation buffer. Sci Rep 11:13453

    Article  CAS  Google Scholar 

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Acknowledgements

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.

Funding

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

  1. Marine Biotechnology, Fish Nutrition and Health Division, ICAR-Central Marine Fisheries Research Institute (CMFRI), Post Box No. 1603, Ernakulam North P.O, Kochi, 682 018, India

    T. G. Sumithra, S. R. Krupesha Sharma, S. Gayathri, Sanal Ebeneezar, K. J. Reshma, P. Vijayagopal & A. Gopalakrishnan

  2. Mandapam Regional Centre of ICAR-CMFRI, Marine Fisheries P.O, 623 520, Mandapam Camp, India

    K. K. Anikuttan, G. Iyyapparaja Narasimapallavan, P. Rameshkumar, M. Sakthivel & G. Tamilmani

  3. Tuticorin Regional Station of ICAR-CMFRI, South Beach Road, 628 001, Tuticorin, India

    D. Linga Prabu

Authors
  1. T. G. Sumithra
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  2. S. R. Krupesha Sharma
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  3. S. Gayathri
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  4. Sanal Ebeneezar
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  6. K. K. Anikuttan
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  7. G. Iyyapparaja Narasimapallavan
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  9. M. Sakthivel
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  10. D. Linga Prabu
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  13. A. Gopalakrishnan
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Contributions

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.

Corresponding author

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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The manuscript has been read and approved for publication by all listed authors.

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The authors declare no competing interests.

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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). https://doi.org/10.1007/s00253-022-12026-6

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  • DOI: https://doi.org/10.1007/s00253-022-12026-6

Keywords

  • Cobia
  • Larvae
  • Metagenomics
  • Preservation
  • Absolute ethanol