Native and exotic oysters in Brazil: Comparative tolerance to hypercapnia.

Environmental hypercapnia in shallow coastal marine ecosystems can be exacerbated by increasing levels of atmospheric CO2. In these ecosystems organisms are expected to become increasingly subjected to pCO2 levels several times higher than those inhabiting ocean waters (e.g.: 10,000µatm), but still our current understanding on different species capacity to respond to such levels of hypercapnia is limited. Oysters are among the most important foundation species inhabiting these coastal ecosystems, although natural oyster banks are increasingly threatened worldwide. In the present study we studied the effects of hypercapnia on two important oyster species, the pacific oyster C. gigas and the mangrove oyster C. brasiliana, to bring new insights on different species response mechanisms towards three hypercapnic levels (ca. 1,000; 4,000; 10,000 µatm), by study of a set of biomarkers related to metabolic potential (electron transport system - ETS), antioxidant capacity (SOD, CAT, GSH), cellular damage (LPO) and energetic fitness (GLY), in two life stages (juvenile and adult) after 28 days of exposure. Results showed marked differences between each species tolerance capacity to hypercapnia, with contrasting metabolic readjustment strategies (ETS), different antioxidant response capacities (SOD, CAT, GSH), which generally allowed to prevent increased cellular damage (LPO) and energetic impairment (GLY) in both species. Juveniles were more responsive to hypercapnia stress in both congeners, and are likely to be most sensitive to extreme hypercapnia in the environment. Juvenile C. gigas presented more pronounced biochemical alterations at intermediate hypercapnia (4,000µatm) than C. brasiliana. Adult C. gigas showed biochemical alterations mostly in response to high hypercapnia (10,000µatm), while adult C. brasiliana were less responsive to this environmental stressor, despite presenting decreased metabolic potential. Our data bring new insights on the biochemical performance of two important oyster species, and suggest that the duration of extreme hypercapnia events in the ecosystem may pose increased challenges for these organisms as their tolerance capacity may be time limited.

Biochemical alterations in native and exotic oyster species in Brazil in response to increasing temperature.

The increase of temperature in marine coastal ecosystems due to atmospheric greenhouse gas emissions is becoming an increasing threat for biodiversity worldwide, and may affect organisms' biochemical performance, often resulting in biogeographical shifts of species distribution. At the same time, the introduction of non-native species into aquatic systems also threatens biodiversity and ecosystem functions. Oysters are among the most valuable socio economic group of bivalve species in global fishery landings, and also provide numerous ecosystem services. However, the introduction of non-native oyster species, namely Crassostrea gigas for aquaculture purposes may threaten native oyster species, mainly by out competing their native congeners. It is therefore of upmost importance to understand physiological and biochemical responses of native and introduced oyster species in a scenario of global temperature rise, in order to provide knowledge that may allow for better species management. Hence, we compared biochemical alterations of the introduced C. gigas and the native Crassostrea brasiliana, the most important oyster species in Brazil, in response to different thermal regimes for 28days (24, 28 and 32°C). For this, metabolism (ETS), energy content (GLY), antioxidant system (SOD, CAT and GSH/GSSG) and cellular damage (LPO) were assessed in adult and juvenile specimens of both species. Juvenile C. gigas were the most affected by increased temperatures, presenting higher mortality, more pronounced antioxidant response (SOD), whereas adults were more tolerant than juveniles, showing no mortality, no significant changes in antioxidant enzymes activity neither energy expenditure. Native C. brasiliana juveniles presented lower mortality and less pronounced biochemical alterations were noted at higher temperature comparing to non-native C. gigas juveniles. Adult C. brasiliana were the least responsive to tested temperatures. Results obtained in this study bring interesting new insights on different oyster species life stages' physiological and biochemical tolerance towards thermal stress. The native species C. brasiliana showed ability to maintain biochemical performance at higher temperatures, with less pronounced biochemical changes than the non-native species. The introduced (C. gigas) showed to be more sensitive, presenting biochemical alterations to cope with the increase of temperature. Despite the lower observed fitness of the introduced species to temperatures closer to those naturally experienced by the native species, the ability of C. gigas to cope with higher temperatures should still raise concerns towards the native species C. brasiliana management and protection.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors.

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.