Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that 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. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Tab.1: Total estimated muskox numbers and quotas in management zones of the Northwest Territories and Nunavut

In view of the drastic growth in the Canadian Inuit population, the rising costs of living, the missing job and income alternatives and the high unemployment rate in the arctic, efforts are being made to make use of the muskox populations in order to provide additional sources of food and/or revenue. The present paper attempts to review the course of muskox utilization in the Canadian Arctic and to tentatively assess its present as weIl as its future economic importance. Starting with the pre-European status of muskoxen in Canada, the drastic reduction in numbers resulting from the combined efforts of hide traders, whalers and expedition parties in the 19th and early 20th centuries, the impact of the legal protection and the recovery since 1917 are being described. Establishing muskox farms with semi-domesticated herds failed in Canada in the 1970's. Since 1969, though, increasing numbers of animals have been allotted to many Inuit communities, and despite the fact that most of the animals were primarily used for subsistence purposes, some communities could reserve part of their quotas for trophy (sport) hunters. While controlled sustainable subsistence and trophy hunts may eventually be carried out over the whole muskox range, including recently colonized northern Quebec, commercial harvesting for meat, hides and wool, introduced in 1981, will at least for some time be restricted to Banks and Victoria islands which at present show 78 % of the Canadian muskox population and 94 % of the overall quota.