Are tetrazolium salts a reliable proxy to asses in vivo respiratory activity in natural marine communities?

Abstract

Recently, the in vivo enzymatic ETS assay, which measures the reduction of INT to INT-formazan in living organisms, has been proposed as a more reliable, proxy of in situ respiration in aquatic ecosystems than the in vitro ETS assay, which requires measuring the reduction of INT in homogenizes, saturated with natural substrates (NADH, NADPH and succinate). Although at first sight the in vivo assay seems to be faster, simpler and more reliable in addressing actual respiration rates, there are some methodological issues that question its usefulness. Here we test the toxicity of INT at three different concentrations (0.1, 0.2 and 0.4mM), during a typical in vivo ETS assay with natural planktonic communities collected in coastal waters of Gran Canaria. Our preliminary results reveal a significant toxic effect of INT over autotrophic and heterotrophic microorganisms. Synechococcus-like cyanobacteria and Picoeukaryotes showed a marked effect in the reduction of the chlorophyll fluorescence, matching the progressive assimilation of INT during the first 6 h of incubation. On the other hand, bacterial viability tests indicated that, just after 10 minutes of incubation, a loss in the cellular membrane integrity occurred in all samples inoculated with INT, regardless of the concentration employed. Although the in vivo enzymatic ETS assay has been presented as a reliable alternative to estimate “actual” respiration, the damaging effect observed over the cellular physiological state due to INT suggests that this method may yield misleading results.