title: Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques.
abstract: BACKGROUND
  Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. Most analytical work uses tissue homogenates, and lacks spatial information on the tissue specific sites of actual ROS formation. Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes at cellular level.
  
  
  RESULTS
  Together with oxidative stress measurements, here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of ROS and RNS formation in the living tissue. Our results indicate that a) mitochondria located in the basal parts of the epithelial cells close to the blood vessels are hyperpolarized with high Δψm, whereas b) the peripheral mitochondria close to the cilia have low (depolarized) Δψm. These mitochondria are densely packed (mitotracker Deep Red 633 staining), have acidic pH (Ageladine-A) and collocate with high formation of nitric oxide (DAF-2DA staining). NO formation is also observed in the endothelial cells surrounding the filament blood sinus. ROS (namely H2O2, HOO(•) and ONOO(-) radicals, assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O2 (•-) than the rest of the tissue. Whether this O2 (•-) production is instrumental to mucus formation or serves antimicrobial protection of the gill surface is unknown. Cells of the ventral bends contain the superoxide forming mucocytes and show significantly higher protein carbonyl formation than the rest of the gill tissue.
  
  
  CONCLUSIONS
  In summary, ROS and RNS formation is highly compartmentalized in bivalve gills under unstressed conditions. The main mechanisms are the differentiation of mitochondria membrane potential and basal ROS formation in inner and outer filament layers, as well as potentially antimicrobial ROS formation in the central blood vessel. Our results provide new insight into this subject and highlight the fact that studying ROS formation in tissue homogenates may not be adequate to understand the underlying mechanism in complex tissues.
authors: Present address: UMR 9190 MARBEC, Groupe fonctionnel AEO, Bat. 24. CC092, Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier, France.
authors: Georgina A
authors: GA
authors: Rivera-Ingraham
authors: Present address: Laboratorio de Ecotoxicología Acuática, INIBIOMA, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-COMAHUE), CEAN, Junín de los Andes, Neuquén, Argentina.
authors: Iara
authors: I
authors: Rocchetta
authors: Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung, Department of Biosciences, Am Handelshafen 12, 27570 Bremerhaven, Germany.
authors: Ulf
authors: U
authors: Bickmeyer
authors: Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung, Department of Biosciences, Am Handelshafen 12, 27570 Bremerhaven, Germany.
authors: Stefanie
authors: S
authors: Meyer
authors: Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung, Department of Biosciences, Am Handelshafen 12, 27570 Bremerhaven, Germany.
authors: Doris
authors: D
authors: Abele
keywords: Bivalve; Fluorescence; Gill; Live-imaging; Mitochondria; RNS; ROS