Comparative 16SrDNA Gene-Based Microbiota Profiles of the Pacific Oyster (Crassostrea gigas) and the Mediterranean Mussel (Mytilus galloprovincialis) from a Shellfish Farm (Ligurian Sea, Italy)

Abstract

The pacific oyster Crassostrea gigas and the Mediterranean mussel Mytilus galloprovincialis are two widely farmed bivalve species which show contrasting behaviour in relation to microbial diseases, with C. gigas being more susceptible and M. galloprovincialis being generally resistant. In a recent study, we showed that different susceptibility to infection exhibited by these two bivalve species may depend on their different capability to kill invading pathogens (e.g., Vibrio spp.) through the action of haemolymph components. Specific microbial-host interactions may also impact bivalve microbiome structure and further influence susceptibility/resistance to microbial diseases. To further investigate this concept, a comparative study of haemolymph and digestive gland 16SrDNA gene-based bacterial microbiota profiles in C. gigas and M. galloprovincialis co-cultivated at the same aquaculture site was carried out using pyrosequencing. Bacterial communities associated with bivalve tissues (hemolymph and digestive gland) were significantly different from those of seawater, and were dominated by relatively few genera such as Vibrio and Pseudoalteromonas. In general, Vibrio accounted for a larger fraction of the microbiota in C. gigas (on average 1.7-fold in the haemolymph) compared to M. galloprovincialis, suggesting that C. gigas may provide better conditions for survival for these bacteria, including potential pathogenic species such as V. aestuarianus. Vibrios appeared to be important members of C. gigas and M. galloprovincialis microbiota and might play a contrasting role in health and disease of bivalve species. Accordingly, microbiome analyses performed on bivalve specimens subjected to commercial depuration highlighted the ineffectiveness of such practice in removing Vibrio species from bivalve tissues.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3

References

  1. 1.

    Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. (2012) Human gut microbiome viewed across age and geography. Nature 486:222–227

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Yarza P, Yilmaz P, Pruesse E, Glockner FO, Ludwig W, Schleifer K-H, et al. (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12:635–645

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Lederberg J, McCray AT (2001) ‘Ome sweet’ omics – a genealogical treasury of words. The. Scientist 15:8

    Google Scholar 

  4. 4.

    Sweet MJ, Bulling MT (2017) On the importance of the microbiome and Pathobiome in coral health and disease. Front Mar Sci. doi:10.3389/fmars.2017.00009

  5. 5.

    Olson JB, Kellogg CA (2010) Microbial ecology of corals, sponges, and algae in mesophotic coral environments. FEMS Microbiol Ecol 73(1):17–30

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Vezzulli L, Pezzati E, Huete-Stauffer C, Pruzzo C, Cerrano C (2013) 16SrDNA pyrosequencing of the Mediterranean gorgonian Paramuricea Clavata reveals a link among alterations in bacterial Holobiont members, anthropogenic influence and disease outbreaks. PLoS One 8(6):e67745

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lokmer A, Wegner KM (2015) Hemolymph microbiome of Pacific oysters in response to temperature, temperature stress and infection. ISME J 9:670–682

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Garnier M, Labreuche Y, Garcia C, Robert M, Nicolas JL (2007) Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas. Microb. Ecol. 53:187–196

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Wendling CC, Batista FM, Wegner MK (2014) Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from Pacific oyster hemolymph. PLoS One 9:e94256

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    King GM, Judd C, Kuske CR, Smith C (2012) Analysis of stomach and gut microbiomes of the eastern oyster (Crassostrea virginica) from coastal Louisiana, USA. PLoS One 7:e51475

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Trabal Fernandez N, Mazon-Suastegui JM, Vazquez-Juarez R, Ascencio-Valle F, Romero J (2013) Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) during commercial production. FEMS Microbiol Ecol 88:69–83

    Article  Google Scholar 

  12. 12.

    Wegner KM, Volkenborn N, Peter H, Eiler A (2013) Disturbance induced decoupling between host genetics and composition of the associated microbiome. BMC Microbiol 13:252

    Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Lokmer A, Kuenzel S, Baines JF, Wegner KM (2016) The role of tissue-specific microbiota in initial establishment success of Pacific oysters. Environ Microbiol 18(3):970–987

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Samain JF, McCombie H (eds.) (2008) Summer mortality of Pacific oyster Crassostrea gigas, the Morest project. Ifremer/Quæ Éditions, Versailles,

    Google Scholar 

  15. 15.

    Pernet F, Lagarde F, Le Gall P, D’Orbcastel ER (2014) Associations between farming practices and disease mortality of Pacific oyster Crassostrea gigas in a Mediterranean lagoon. Aquac Environ Interact 5:99–106

    Article  Google Scholar 

  16. 16.

    Labreuche Y, Soudant P, Goncalves M, Lambert C, Nicolas JL (2006) Effects of extracellular products from the pathogenic vibrio aestuarianus strain 01/32 on lethality and cellular immune responses of the oyster Crassostrea gigas. Dev Comp Immunol 30:367e79

    Article  Google Scholar 

  17. 17.

    Craft JA, Gilbert JA, Temperton B, Dempsey KE, Ashelford K, Tiwari B, et al. (2010) Pyrosequencing of Mytilus Galloprovincialis cDNAs: tissue-specific expression patterns. PLoS One 5(1):e8875

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Pezzati E, Canesi L, Damonte G, Salis A, Marsano F, Grande C, et al. (2015) Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus hemolymph: identification of a serum opsonin involved in mannose-sensitive interactions. Environ Microbiol 17(11):4271–4279

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Canesi L, Grande C, Pezzati E, Balbi T, Vezzulli L, Pruzzo C (2016) Killing of Vibrio cholerae and Escherichia coli strains carrying D-mannose-sensitive ligands by Mytilus hemocytes is promoted by a multifunctional hemolymph serum protein. Microb Ecol 72(4):759–762

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Penders J, Vink C, Driessen C, London N, ThiisC SEE (2005) Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in fecal samples of breast-fed and formula-fed infants by real time PCR. FEMS Microbiol Ecol 24:141–147

    Article  Google Scholar 

  21. 21.

    Luna GM, Dell'Anno A, Pietrangeli B, Danovaro R (2012) A new molecular approach based on qPCR for the quantification of fecal bacteria in contaminated marine sediments. 157(4):446–453

  22. 22.

    IFREMER report (2013) Vibrio splendidus et V. aestuarianus detection by real time polymerase chain reaction. European union reference laboratory for molluscs diseases. Edition n 1, Laboratoire de Génétique et Pathologie des Mollusques Marins, Av. de Mus de Loup, 17390 La Tremblade France. URL http://www.eurlmollusc.eu/content/download/72924/948279/file/Vsplendidus&aestuarianus%20_RealTimePCR.pdf

  23. 23.

    Wilson B, Muirhead A, Bazanella M, Huete-Stauffer C, Vezzulli L, Bourne DG (2013) An improved detection and quantification method for the coral PathogenVibrio coralliilyticus. PLoS One 812:e81800

    Article  Google Scholar 

  24. 24.

    Vezzulli L, Stauder M, Grande C, Pezzati E, Verheye HM, Owens NJP, et al. (2015) gbpA as a novel qPCR target for the species-specific detection of vibrio cholerae O1, O139, non-O1/non-O139 in environmental, stool, and historical continuous plankton recorder samples. PLoS One 10(4):e0123983

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Campbell MS, Wright AC (2003) Real-time PCR analysis of Vibrio vulnificus from oysters. Appl Environ Microbiol 69(12):7137–7144

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, DePaola A (2007) Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl Environ Microbiol 73(18):5840–5847

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR (2006) Microbial diversity in the deep sea and the underexplored ‘rare biosphere’. Proc Natl Acad Sci U S A 103:12115–12120

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Prieur D, Nicolas JL, Plusquellec A, Vigneulle M (1990) Interactions between bivalve mollusks and bacteria in the marine-environment. Oceanogr Mar Biol 28:277–352

    Google Scholar 

  29. 29.

    Olafsen JA, Mikkelsen HV, Glaever HM, Hansen GH (1993) Indigenous bacteria in hemolymph and tissues of marine bivalves at low-temperatures. Appl Environ Microbiol 59:1848–1854

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Pruzzo C, Gallo G, Canesi L (2005) Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. Environ Microbiol 7(6):761–772

    Article  PubMed  Google Scholar 

  31. 31.

    Holmström C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30(4):285–293

    Article  PubMed  Google Scholar 

  32. 32.

    Engel S, Jensen PR, Fenical W (2002) Chemical ecology of marine microbial defense. J Chem Ecol 28(10):1971–1985

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Balbi T, Fabbri R, Cortese K, Smerilli A, Ciacci C, Grande C, et al. (2013) Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32. Fish Shellfish Immunol 35(6):1906–1915

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Bayne BL, Bayne CJ, Carefoot TC, Thompson RJ (1976) The physiological ecology of Mytilus californianus Conrad. 1. Metabolism and energy balance. Oecologia 22:211–228

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Matias PM, Pereira IAC, Soares CM, Carrondo MA (2005) Sulphate respiration from hydrogen in Desulfovibrio bacteria: a structural biology overview. Prog Biophys Mol Biol 89:292–329

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Wang WX, Widdows J (1993) Metabolic responses of the common mussel Mytilus edulis to hypoxia and anoxia. Mar Ecol Prog Ser 95:205–214

    Article  Google Scholar 

  37. 37.

    Lee R, Lovatelli A, Ababouch L (2008) Bivalve depuration: fundamental and practical aspects. FAO Fisheries Technical Paper. No. 511. FAO, Rome, p 139

  38. 38.

    Di Cesare A, Eckert EM, Teruggi A, Fontaneto D, Bertoni R, Callieri C, et al. (2015) Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake. Mol Ecol 24(15):3888–3900

    Article  PubMed  Google Scholar 

  39. 39.

    Di Cesare A, Pasquaroli S, Vignaroli C, Paroncini P, Luna GM, Manso E, et al. (2014) The marine environment as a reservoir of enterococci carrying resistance and virulence genes strongly associated with clinical strains. Environ Microbiol Rep 6(2):184–190

    Article  PubMed  Google Scholar 

  40. 40.

    Neave MJ, Apprill A, Ferrier-Pagès C, Voolstra CR (2016) Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas. Appl Microbiol Biotechnol 100(19):8315–8324

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Chauhan A, Green S, Pathak A, Thomas J, Venkatramananc R (2013) Whole-genome sequences of five oyster-associated bacteria show potential for crude oil hydrocarbon degradation. Genome Announc 1(5):e00802–e00813

    Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Richards GP (2014) Bacteriophage remediation of bacterial pathogens in aquaculture: a review of the technology. Bacteriophage 4(4):e975540

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We wish to thank the director and staff of Coop. Mitilicoltori Spezzini A.R.L. (La Spezia, Italy) for their invaluable collaboration during the sampling activity and for kindly allowing the present study. We are particularly indebted to Prof. Luigi Pane and Dr. Guido Bonello (University of Genoa) for helpful assistance during bivalve sampling. We are also kindly grateful to Dr. Adriana Amaro (University of Genoa) for precious help with pyrosequencing analysis. This work was supported by the HORIZON2020 project “Preventing and mitigating farmed bivalve disease—VIVALDI (grant number 678589)”.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Luigi Vezzulli.

Electronic Supplementary Material

Table S1

(DOCX 16 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Vezzulli, L., Stagnaro, L., Grande, C. et al. Comparative 16SrDNA Gene-Based Microbiota Profiles of the Pacific Oyster (Crassostrea gigas) and the Mediterranean Mussel (Mytilus galloprovincialis) from a Shellfish Farm (Ligurian Sea, Italy). Microb Ecol 75, 495–504 (2018). https://doi.org/10.1007/s00248-017-1051-6

Download citation

Keywords

  • Mytilus galloprovincialis
  • Crassostrea gigas
  • Next generation sequencing
  • 16SrDNA
  • Microbiota