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Invertebrate Adaptations to Salinity changes in the Intertidal environment: a Life-imaging approach

Final Report Summary - IAS-LIFE (Invertebrate Adaptations to Salinity changes in the Intertidal environment: a Life-imaging approach)

Intertidal zoobenthic organisms inhabit one of the most challenging habitats of the world, since they live at the margins of the marine and the terrestrial realms. Animals inhabiting these areas must daily cope with the most extreme conditions of both aerial and aquatic regimes. Regular tides or random meteorological events may cause drastic environmental variations. Exposed organisms provide a unique perspective on the wide variety of evolutionary ecophysiological osmoregulation mechanisms that allow them to colonize this challenging habitat. In the context of global climate change, understanding the ecophysiological adaptation of organisms is essential for realistically evaluating the impact that changes in environmental conditions will have on organisms and ecosystems.

Dealing with stress usually requires energetic expenses to fuel acclimation processes. Mitochondria, as the main suppliers of such energy requirements, commonly produce free radicals (FR), reactive-oxygen species (ROS) and reactive-nitrogen species (RNS) that occur in aerobically functioning cells as by-products of cellular respiration and of several cellular defense and detoxification mechanisms. Accumulation of FR that cannot be detoxified by the cellular antioxidant system leads to “oxidative stress” (OS), which is directly linked with organismal fitness and cellular/organismal senescence. Thus, studying the balance between energy requirements, FR production and antioxidant defense arises as a highly relevant approach to study acclimation to environmental changes.

The aim of the “IAS-Life” project is to contribute filling the important gap of knowledge on how intertidal invertebrate species can physiologically handle such environmental changes in terms of FR production that derives from exposure to salinity shocks. More specifically, this project aims, not only to characterize the spatio-temporal generation of FR, but to explore if these harmful molecules, in small quantities, have indeed a role in inducing physiological acclimation to environmental stress, namely salinity shocks.

“IAS-Life” project started in 2014 thanks to the the funding of the European Commission through a Marie-Curie IEF fellowship awarded to Dr. Rivera-Ingraham and under the scientific supervision of Prof. Lignot. Given that most of the studies approaching this subject work in-vito or ex-vivo, the challenge undertaken by this project was to work in-vivo, aiming to have a better understanding of the complexity of intercellular signaling processes leading to salinity acclimation. Thus, along other intertidal invertebrates such as the green crab Carcinus aestuarii, the small and transparent flatworm Macrostomum lignano was the focus of “IAS-Life”. This marine meiofaunal platyhelminth allows the application of live-imaging techniques which, through the use of specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, allows analyzing physiological processes in-vivo.

The two intertidal invertebrates used in this project were demonstrated to be osmo-conforming in seawater and hypersaline environments. However, at lower environmental salinities, these species behaved as osmo-regulators, maintaining their internal body fluids or intracellular medium at higher osmolalities compared to their environment. Despite this common behavior, the crab and the flatworm models responded quite differently in terms of redox balance and energy requirements: while under lowsanility platyhelminths go into metabolic depression, crabs increase their overall respiration rates and energy expenditures. However, in both cases, organisms increase their antioxidant defences. But if flatworms at low salinity are indeed facing metabolic depression, what is the biological role of an increase in antioxidants and how is this upregulation and the hyper-osmoregulatory mechanisms fuelled under conditions of restricted energy production? These results allowed hypothesizing that this increase in antioxidants could be a “preparation for oxidative stress”, i.e. a mechanism to counteract the production of free radicals upon returning to seawater.
But in this acclimation process, are FR merely deleterious molecules or are they implicated in stress signaling and homeostasis? To answer this question, the redox status in both models was experimentally altered by treating animals with an antioxidant solution, and after exposed to a low salinity shock. The antioxidant treatment prevented antioxidant upregulation, which demonstrates that FR are actually mediating the redox response. For the specific case of crabs, this antioxidant pre-treatment protected non-osmoregulatory tissues such as anterior gills from the FR production induced by hyposmotic shock and allows these tissues to increase expression osmoregulation-involved membrane pumps.
Taken together, the data resulting from the “IAS-Life” project, have allowed developing a functional model explaining how reactive oxygen species are involved in the physiological behavior of intertidal organisms during hyper-osmoregulation in dSW.

The results of the “IAS-Life” project reinforce the hypothesis that, for specific organisms or certain tissues, small amounts of FR are required for the correct acclimation of organisms to salinity change. Taken together, this understanding will prove of high relevance in disentangling the mechanistic pathways of acclimation. This knowledge has the potential to impact several and widely diverse fields of study. Agronomical methods have, for example, the potential to apply this information, which could contribute to the development of new protocols aiming to increase animal tolerance to stress through natural and sustainable ways leading to decrease the use of chemical or antibiotics for the same purposes.