Membrane vectorial lipidomic features of coral host cells' plasma membrane and lipid profiles of their endosymbionts Cladocopium.

The symbiotic relationships between coral animal host and autotrophic dinoflagellates are based on the mutual exchange and tight control of nutritional inputs supporting successful growth. The corals Sinularia heterospiculata and Acropora aspera were cultivated using a flow-through circulation system supplying seawater during cold and warm seasons of the year, then sorted into host cells and symbionts and subjected to phylogenetic, morphological, and advanced lipid analyses. Here we show, that the lipidomes of the dinoflagellates Cladocopium C1/C3 and acroporide-specific Cladocopium hosted by the corals, are determined by lipidomic features of different thermosensitivity and unique betaine- and phospholipid molecular species. Phosphatidylserines and ceramiaminoethylphosphonates are not detected in the symbionts and predominantly localized on the inner leaflet of the S. heterospiculata host plasma membrane. The transmembrane distribution of phosphatidylethanolamines of S. heterospiculata host changes during different seasons of the year, possibly contributing to mutualistic nutritional exchange across this membrane complex to provide the host with a secure adaptive mechanism and ecological benefits.

Physiological processes and lipidome dynamics in the soft coral Sinularia heterospiculata under experimental bleaching.

Coral polyps host intracellular symbiotic dinoflagellates (SD). The loss of SD (referred as bleaching) under stressful environmental conditions is the main reason of coral reef destruction, and therefore, intensively studied over the world. Lipids are the structural base of biomembranes and energy reserve of corals and are directly involved in the coral bleaching. In order to establish a relationship between coral tissue morphology, physiological processes and lipidome dynamics during bleaching, the soft coral Sinularia heterospiculata was exposed to experimental heat stress (33 °C) for 72 h. A chlorophyll content, structure of cells, the level of reactive oxygen species (ROS), and molecular species of storage and structural lipids were analyzed. After 24 h of heat exposure, the level of ROS-positive SD cells did not increase, but the host tissues lost a significant part of SD. The removal of SD cells by exocytosis were suggested. Exocytosis was presumed to prevail at earlier stages of the soft coral bleaching. Symbiophagosomes with degenerative SD were observed in the stressed coral host cells. After 24 h, the content of phosphatidylinositols, which involved in apoptosis and autophagy, was significantly decreased. The innate immune response was triggered, and SD were digested by the coral host. After 48 h, a degradation of SD chloroplasts and a decrease in the specific monogalactosyldiacylglycerol molecular species were detected that confirmed a disruption of lipid biosynthesis in chloroplasts. At the end of coral bleaching, the appearance of oxidized phosphatidylethanolamines, indicating damage to the host membranes, and the degradation of the coral tissues were simultaneously observed. Thus, a switch between dominant mechanisms of the SD loss during bleaching of S. heterospiculata was found and proved by certain variations of the lipidomic profile. Lipidomic parameters may become indicators of physiological processes occurring in the symbiotic coral organism and may be used for assessing anthropogenic or natural destructive effects on coral reefs.

Isolation, characterization, and ecotoxicological application of marine mammal skin fibroblast cultures.

Marine mammal cell cultures are a multifunctional instrument for acquiring knowledge about life in the world's oceans in physiological, biochemical, genetic, and ecotoxicological aspects. We succeeded in isolation, cultivation, and characterization of skin fibroblast cultures from five marine mammal species. The cells of the spotted seal (Phoca largha), the sea lion (Eumetopias jubatus), and the walrus (Odobenus rosmarus) are unpretentious to the isolation procedure. The sea otter (Enhydra lutris) fibroblasts should be isolated by trypsin disaggregation, while only mechanical disaggregation was suitable for the beluga whale (Delphinapterus leucas) cells. The cell growth parameters have been determined allowing us to find the optimal seeding density for continuous and effective cultivation. The effects of nonpathogenic algal extracts on proliferation, viability, and functional activity of marine mammal cells in vitro have been presented and discussed for the first time.

The death pathways in mussel larval cells after a freeze-thaw cycle.

We analyzed cell viability, caspase activity, plasma membrane alterations and cell ultrastructure morphology to estimate the morphological and biochemical alterations that occur in bivalve molluscan cell cultures during cryopreservation. The use of 5% dymethyl sulfoxide as a cryoprotectant resulted in greater cell survival and a scarcity of destroyed cells lacking cytosol among dead cells. In this case, almost all cells died through necrosis or apoptosis, which appeared to increase in mussel cell cultures after a freeze-thaw cycle. Apoptosis was not a main death pathway in mussel cells, but it was induced in a significant part of these cells (up to 24%) immediately after thawing and depended mostly on the cryoprotectant used. Regardless of the type of the used cryoprotectant, we observed some nuclear aberrations in cells after freezing-thawing, such as few multipolar mitoses or the absence of a division spindle in mitotic cells. After analyzing different methods for assessing cell damage, the best results were obtained from optimal approaches that could provide information regarding the cell disruption level after freezing-thawing and could be considered for future studies.

The contribution of apoptosis and necrosis in freezing injury of sea urchin embryonic cells.

Sea urchins have recently been reported to be a promising tool for investigations of oxidative stress, UV light perturbations and senescence. However, few available data describe the pathway of cell death that occurs in sea urchin embryonic cells after cryopreservation. Our study is focused on the morphological and functional alterations that occur in cells of these animals during the induction of different cell death pathways in response to cold injury. To estimate the effect of cryopreservation on sea urchin cell cultures and identify the involved cell death pathways, we analyzed cell viability (via trypan blue exclusion test, MTT assay and DAPI staining), caspase activity (via flow cytometry and spectrophotometry), the level of apoptosis (via annexin V-FITC staining), and cell ultrastructure alterations (via transmission electron microscopy). Using general caspase detection, we found that the level of caspase activity was low in unfrozen control cells, whereas the number of apoptotic cells with activated caspases rose after freezing-thawing depending on cryoprotectants used, also as the number of dead cells and cells in a late apoptosis. The data using annexin V-binding assay revealed a very high apoptosis level in all tested samples, even in unfrozen cells (about 66%). Thus, annexin V assay appears to be unsuitable for sea urchin embryonic cells. Typical necrotic cells with damaged mitochondria were not detected after freezing in sea urchin cell cultures. Our results assume that physical cell disruption but not freezing-induced apoptosis or necrosis is the predominant reason of cell death in sea urchin cultures after freezing-thawing with any cryoprotectant combination.

Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).