INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure


Autoři: E. Elena García-Martín aff001;  Isabel Seguro aff001;  Carol Robinson aff001
Působiště autorů: Centre for Ocean and Atmospheric Sciences, School of Environmental Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom aff001
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225954

Souhrn

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

Klíčová slova:

Cell walls – Dissolved oxygen – Eukaryota – Oxygen – Oxygen consumption – Plankton – Respiration


Zdroje

1. Dufour P, Colon M. The tetrazolium reduction method for assessing the viability of individual bacterial cells in aquatic environments: improvements, performance and applications. Hydrobiologia. 1992; 232(3):211–8.

2. Stellmach J, Severin E. A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt—formazan reaction of Ehrlich ascites tumour cells. The Histochemical Journal. 1987; 19(1):21–6. doi: 10.1007/bf01675289 3583812

3. del Giorgio PA, Prairie Y, Bird D. Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry. Microbial Ecology. 1997; 34(2):144–54. doi: 10.1007/s002489900044 9230102

4. Packard TT. The measurement of respiratory electron transport activity in marine phytoplankton Journal of Marine Research. 1971; 29(3):235–44.

5. Hatzinger PB, Palmer P, Smith RL, Peñarrieta CT, Yoshinari T. Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria. Journal of Microbiological Methods. 2003; 52(1):47–58. doi: 10.1016/s0167-7012(02)00132-x 12401226

6. García-Martín EE, Aranguren-Gassis M, Karl DM, Martínez-García S, Robinson C, Serret P, et al. Validation of the in vivo iodo-nitro-tetrazolium (INT) salt reduction method as a proxy for plankton respiration. Frontiers in Marine Science. 2019; 6(220).

7. Martínez-García S, Fernández E, Aranguren-Gassis M, Teira E. In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities. Limnology and Oceanography: methods. 2009; 7:459–69.

8. García-Martín EE, Aranguren-Gassis M, Hartmann M, Zubkov MV, Serret P. Contribution of bacterial respiration to plankton respiration from 50°N to 44°S in the Atlantic Ocean. Progress in Oceanography. 2017; 158:99–108.

9. Aranguren-Gassis M, Teira E, Serret P, Martínez-García S, Fernández E. Potential overestimation of bacterial respiration rates in oligotrophic plankton communities. Marine Ecology Progress Series. 2012; 453:1–10.

10. Martínez-García S, Karl DM. Microbial respiration in the euphotic zone at Station ALOHA. Limnology and Oceanography. 2015; 60(3):1039–50.

11. Villegas-Mendoza J, Cajal-Medrano R, Helmut M. The chemical transformation of the cellular toxin INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(phenyl) tetrazolium chloride) as an indicator of prior respiratory activity in aquatic bacteria. International Journal of Molecular Sciences. 2019; 20(3):782.

12. Villegas-Mendoza J, Cajal-Medrano R, Maske H. 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. Microbial Ecology. 2015; 70(4):1004–11. doi: 10.1007/s00248-015-0626-3 25991603

13. Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Applied and Environmental Microbiology. 1992; 58(6):1801–8. 1622256

14. Trevors JT, Mayfield CI, Inniss WE. Measurement of electron transport system (ETS) activity in soil. Microbial Ecology. 1982; 8(2):163–8. doi: 10.1007/BF02010449 24225810

15. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review. 2005; 11:127–52. doi: 10.1016/S1387-2656(05)11004-7 16216776

16. Nachlas MM, Margulies SI, Seligman AM. Sites of electron transfer to tetrazolium salts in the succinoxidase system. The Journal of Biological Chemistry. 1960; 235(9):2737–43.

17. Hudson RJ, Morel FM. Trace metal transport by marine microorganisms: implications of metal coordination kinetics. Deep Sea Research Part I: Oceanographic Research Papers. 1993; 40(1):129–50.

18. Syrett P, Thomas E. The assay of nitrate reductase in whole cells of Chlorella: strain differences and the effect of cell walls. New Phytologist. 1973; 72(6):1307–10.

19. Guillard RR, Ryther JH. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology. 1962; 8(2):229–39.

20. Keller M, Guillard RR. Factors significant to marine dinoflagellate culture. In: Anderson D, White A, Baden D, editors. Toxic dinoflagellates. New York: Elsevier; 1985. p. 113–6.

21. Keller MD, Selvin RC, Claus W, Guillard RR. Media for the culture of oceanic ultraphytoplankton 1, 2. Journal of Phycology. 1987; 23(4):633–8.

22. Carritt DE, Carpenter JH. Comparison and evaluation of currently employed modifications of the Winkler method for determining dissolved oxygen in seawater; a NASCO Report. Journal of Marine Research. 1966; 24:286–319.

23. Clarke MRB. The reduced major axis of a bivariate sample. Biometrika. 1980; 67(2):441–6.

24. Thom SM, Horobin R, Seidler E, Barer M. Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. Journal of Applied Bacteriology. 1993; 74(4):433–43. doi: 10.1111/j.1365-2672.1993.tb05151.x 7683637

25. Tuovila BJ, LaRock PA. Effect of species difference and growth rate in the use of INT as an indicator of bacterial respiration. Journal of Microbiological Methods. 1985; 4(3–4):185–8.

26. Smith J, McFeters G. 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. Journal of Applied Bacteriology. 1996; 80(2):209–15. doi: 10.1111/j.1365-2672.1996.tb03212.x 8642015

27. Smith JJ, McFeters GA. Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2, 3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12. Journal of Microbiological Methods. 1997; 29(3):161–75.

28. Marañón E. Inter-specific scaling of phytoplankton production and cell size in the field. Journal of Plankton Research. 2007; 30(2):157–63.

29. Relexans J. Measurement of the respiratory electron transport system (ETS) activity in marine sediments: state-of-the-art and interpretation. II. Significance of ETS activity data. Marine Ecology Progress Series. 1996; 136:289–301.

30. Filella A, Baños I, Montero MF, Hernández-Hernández N, Rodríguez-Santos A, Ludwig A, et al. Plankton community respiration and ETS activity under variable CO2 and nutrient fertilization during a mesocosm study in the subtropical North Atlantic. Frontiers in Marine Science. 2018; 5:310.

31. Møller IM. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annual Review of Plant Physiology and Plant Molecular Biology. 2001; 52(1):561–91.

32. McBee ME, Chionh YH, Sharaf ML, Ho P, Cai MWL, Dedon PC. Production of superoxide in bacteria is stress-and cell state-dependent: a gating-optimized flow cytometry method that minimizes ROS measurement artifacts with fluorescent dyes. Frontiers in Microbiology 2017; 8:459. doi: 10.3389/fmicb.2017.00459 28377755


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2019 Číslo 12