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INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity

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Abstract

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14–49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

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References

  1. Robinson C, Williams PJLeB (2005) Respiration and its measurement in the surface waters. In: del Giorgio PA, Williams PJLeB (eds.) Respiration in Aquatic Ecosystems. Oxford University Press, New York, NY, pp 147–180

  2. Arístegui J, Agustí S, Middelburg JJ, Duarte CM (2005) Respiration in the mesopelagic and bathypelagic zones of the oceans. In: del Giorgio PA, Williams PJLeB (eds) Respiration in Aquatic Ecosystems. Oxford University Press, New York, pp 181–205

  3. Robinson C (2018) Microbial respiration, the engine of ocean deoxygenation. Front. Mar. Sci. 5(30):533. https://doi.org/10.3389/fmars.2018.00533

    Article  Google Scholar 

  4. Ducklow HW, Doney SC (2013) What is the metabolic state of the oligotrophic ocean? A debate. Annu Nat Rev Mar 5:525–533. https://doi.org/10.1146/annurev-marine-121211-172331

    Article  Google Scholar 

  5. Packard TT (1971) The measurement of respiratory electron transport activity in marine phytoplankton. J. Mar. Res. 29:235–244

    Google Scholar 

  6. Arístegui J, Montero MF (1995) The relationship between community respiration and ETS activity in the ocean. J. Plankton Res. 17(7):1563–1571. https://doi.org/10.1093/plankt/17.7.1563

    Article  Google Scholar 

  7. Filella A, Baños I, Montero MF, Hernández-Hernández N, Rodríguez-Santos A, Ludwig A, Riebesell U, Arístegui J (2018) Plankton community respiration and ETS activity under variable CO2 and nutrient fertilization during a mesocosm study in the subtropical North Atlantic. Front. Mar. Sci. 5:310. https://doi.org/10.3389/fmars.2018.00310

    Article  Google Scholar 

  8. Martínez-García S, Fernández E, Aranguren-Gassis M, Teira E (2009) In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities. Limnol. Oceanogr. Methods 7:459–469. https://doi.org/10.4319/lom.2009.7.459

    Article  Google Scholar 

  9. García-Martin EE, Aranguren-Gassis M, Karl DM, Martínez-García S, Robinson C, Serret P, Teira E (2019) Validation of the in vivo Iodo-nitro-tetrazolium (INT) salt reduction method as a proxy for plankton respiration. Front. Mar. Sci. 6:220. https://doi.org/10.3389/fmars.2019.00220

    Article  Google Scholar 

  10. Aranguren-Gassis M, Teira E, Serret P, Martínez-García S, Fernández E (2012) Potential overestimation of bacterial respiration rates in oligotrophic plankton communities. Mar. Ecol. Prog. Ser. 453:1–10. https://doi.org/10.3354/meps09707

    Article  CAS  Google Scholar 

  11. García-Martín EE, Daniels CJ, Davidson K, Lozano J, Mayers KM, McNeill S, Mitchell E, Poulton AJ, Purdie DA, Tarran GA, Whyte C, Robinson C (2017) Plankton community respiration and bacterial metabolism in a North Atlantic Shelf Sea during spring bloom development (April 2015). Prog. Oceanogr. 177:1-10.https://doi.org/10.1016/j.pocean.2017.11.002

  12. Servais P, Agogué H, Courties C, Joux F, Lebaron P (2001) Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments? FEMS Microbiol. Ecol. 35(2):171–179. https://doi.org/10.1111/j.1574-6941.2001.tb00801.x

    Article  CAS  PubMed  Google Scholar 

  13. Gasol JM, Arístegui J (2007) Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity. Aquat. Microb. Ecol. 46:71–83. https://doi.org/10.3354/ame046071

    Article  Google Scholar 

  14. Grégori G, Citterio S, Ghiani A, Labra M, Sgorbati S, Brown S, Denis M (2001) Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl. Environ. Microbiol. 67(10):4662–4670. https://doi.org/10.1128/AEM.67.10.4662-4670.2001

  15. Falcioni T, Papa S, Gasol JM (2008) Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl Environ Microb 74:1767–1779. https://doi.org/10.1128/AEM.01668-07

    Article  CAS  Google Scholar 

  16. Gasol JM, Zweifel UL, Peters F, Fuhrman JA, Hagström A ̊ (1999) Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol. 65: 4475–4483

  17. Smith DC, Azam F (1992) A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine. Mar Microb Food Webs 6(2):107–114

    Google Scholar 

  18. Pinheiro J, Bates D, DebRoy S, Sarkar D, Core Team R (2018) Nlme: linear and nonlinear mixed effects models. R package version 3:1–137 https://CRAN.R-project.org/package=nlme

    Google Scholar 

  19. R Core Team (2018) R: A language and environment for statistical computing. v3.3.2 https://www.R-project.org/ Vienna, Austria. R Foundation for Statistical Computing

  20. Servais P, Casamayor EO, Courties C, Catala P, Parthuisot N, Lebaron P (2003) Activity and diversity of bacterial cells with high and low nucleic acid content. Aquat. Microb. Ecol. 33:41–51. https://doi.org/10.3354/ame033041

    Article  Google Scholar 

  21. Tuovilla BJ, LaRock PA (1985) Effect of species difference and growth rate in the use of INT as an indicator of bacterial respiration. J Microbiol Meth 4:185–188. https://doi.org/10.1016/0167-7012(85)90035-1

    Article  Google Scholar 

  22. Hatzinger PB, Palmer P, Smith RL, Peñarrieta CT, Yoshinari T (2003) Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria. J Microbiol Meth 52(1):47–58. https://doi.org/10.1016/S0167-7012(02)00132-X

    Article  CAS  Google Scholar 

  23. May PS, Winter JW, Fried GH, Antopol W (1960) Effect of tetrazolium salts on selected bacterial species. Proc. Soc. Exp. Bio. Med 105(2):364–366

    Article  CAS  Google Scholar 

  24. Smith JJ, McFeters GA (1996) Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2, 3-ditolyl tetrazolium chloride) reduction in Escherichia coli. J Appl Bacteriol 80(2):209–215. https://doi.org/10.1111/j.1365-2672.1996.tb03212.x

    Article  CAS  PubMed  Google Scholar 

  25. Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58(6):1801–1808

    Article  CAS  Google Scholar 

  26. Villegas-Mendoza J, Cajal-Medrano R, Maske H (2015) INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(phenyl) tetrazolium chloride) is toxic to prokaryote cells precluding its use with whole cells as a proxy for in vivo respiration. Microb. Ecol. 70:1–8. https://doi.org/10.1007/s00248-015-0626-3

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Josep M. Gasol for his help with the leucine uptake experiments, Ángelo Santana for statistical advice, and Marta Sebastián for thoughtful discussions.

Funding

IB was supported by a FPI fellowship (BES-2016- 078407) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and by an “INNOVA Canarias 2020” grant from the “Fundación Universitaria de Las Palmas de Gran Canaria”. This is a contribution to the FLUXES project (CTM2015-69392-C3-1-R) funded by the Spanish government (Plan Nacional I + D).

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Authors and Affiliations

  1. Instituto de Oceanografía y Cambio Global, IOCAG, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

    Isabel Baños, María F. Montero & Javier Arístegui

  2. Aix-Marseille Université, Université de Toulon, CNRS, IRD, Mediterranean Institute of Oceanography (MIO), UM 110, 13288, Marseille, France

    Mar Benavides

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  1. Isabel Baños
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  2. María F. Montero
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  3. Mar Benavides
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  4. Javier Arístegui
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Correspondence to Javier Arístegui.

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Baños, I., Montero, M.F., Benavides, M. et al. INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity. Microb Ecol 80, 237–242 (2020). https://doi.org/10.1007/s00248-019-01479-4

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  • DOI: https://doi.org/10.1007/s00248-019-01479-4

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