Dessication Stress

The threat of desiccation for organisms inhabiting the intertidal zone occurs during emersion at low tides or when organisms are positioned in the high intertidal zone, where wetting occurs primarily by spring tides, storm waves, and spray. Drying due to evaporative water loss is the most common mechanism for dehydration, although during winter in northern temperate regions freezing can also occur, which reduces the liquid water in extracellular fluids and can lead to intracellular dehydration in multicellular organisms. Freezing tolerance has been reported and characterized for a number of intertidal invertebrates, including gastropods such as an air-breathing snail and a periwinkle, and bivalve genera including the common and ribbed mussels

Effects of Osmotic Stress on Oxygen Consumption of Drosophila Cells (Kc167)

This article investigates the effect of osmotic stress on a drosophila cell line called Kc167. The embryonic-derived fly (Drosophila melanogaster) cell line, Kc-167, was employed as a model for water-stress sensitivity in Arthropods. Like mammalian cells, cells derived from the fruit fly contain the same basic set of membranous components found in all eukaryotic cells. A series of experiments were conducted to characterize the mitochondrial repones of Kc167 cells to water stress. Precisely, the oxygen flux in a sealed respirometer chamber containing Kc167 cells was measured under hyperosmotic and control conditions. Mitochondrial uncouplers were used in some experiments for intact and chemically permeabilized cells to gain detailed information on mitochondrial integrity in response to increased solute concentration. Mitochondria are the primary ATP producer in the cell and consume oxygen in a process termed oxidative phosphorylation. Therefore, oxygen consumption rates can be used to assess the impact of water-limited states on cellular bioenergetics. Conducted experiments were performed to measure the following: • The basal oxygen consumption rates of Kc167 cells. • Consumption under conditions of oxidative stress • The oxygen consumption of chemically permeabilized cells • The max mitochondrial uncoupling that the Kc167 could withstand

Reduced mitochondrial efficiency explains mismatched growth and metabolic rate at supraoptimal temperatures.

The relationship between whole-organism growth and metabolism is generally assumed to be positive and causative; higher metabolic rates support higher growth rates. In Manduca sexta, existing data demonstrate a deviation from this simple prediction: at supraoptimal temperatures for larval growth, metabolic rate keeps increasing while growth rate is decreasing. This mismatch presumably reflects the rising “cost of maintenance” with temperature. Precisely what constitutes this cost is not clear, but we suspect the efficiency with which mitochondria harness oxygen and organic substrates into cellular energy (ATP) is key. We tested this by integrating existing data on M. sexta growth and metabolism with new data on mitochondrial bioenergetics across the temperature range 14°–42°C. Across this range, our measure of mitochondrial efficiency closely paralleled larval growth rates. At supraoptimal temperatures for growth, mitochondrial efficiency was reduced, which could explain the mismatch between growth and metabolism observed at the whole-organism level

Mitochondrial energetics of benthic and pelagic Antarctic teleosts.

Antarctic fauna are highly adapted to the frigid waters of the Southern Ocean. This study describes the in vitro temperature sensitivity of oxygen consumption rates measured in liver mitochondria from the pelagic notothenioid Pleuragramma antarcticum between 5 and 35 C. Oxygen fluxes were measured after the addition of millimolar levels of pyruvate, malate, succinate and glutamate (state II, LEAK) and saturating levels of ADP [state III, oxidative phosphorylation (OXPHOS)]. State III respiration significantly decreased above 18.7 C. A comparison of the oxidative capacities among P. antarcticum and other notothenioids showed significant differences in state III respiration, where benthic species exhibited about 50 % lower rates than P. antarcticum . In addition, state III respiration rates normalized per milligram of mitochondrial protein of P. antarcticum were up to eight times higher than state III rates reported in the literature for other notothenioids. The comparatively high respiration rates measured in this study may be explained by our approach, which engaged both complexes I and II under conditions of oxidative phosphorylation. State III rates of independently activated complexes I and II were found to range from 42 to 100 % of rates obtained when both complexes were activated simultaneously in the same species. The remarkable tolerance of P. antarcticum OXPHOS toward warmer temperatures was unexpected for an Antarctic stenotherm and may indicate that thermal sensitivity of their mitochondria is not the driving force behind their stenothermy

Mitochondrial Energetics of Benthic and Pelagic Antarctic Teleosts

Antarctic fauna are highly adapted to the frigid waters of the Southern Ocean. This study describes the in vitro temperature sensitivity of oxygen consumption rates measured in liver mitochondria from the pelagic notothenioid Pleuragramma antarcticum between 5 and 35 C. Oxygen fluxes were measured after the addition of millimolar levels of pyruvate, malate, succinate and glutamate (state II, LEAK) and saturating levels of ADP [state III, oxidative phosphorylation (OXPHOS)]. State III respiration significantly decreased above 18.7 C. A comparison of the oxidative capacities among P. antarcticum and other notothenioids showed significant differences in state III respiration, where benthic species exhibited about 50 % lower rates than P. antarcticum . In addition, state III respiration rates normalized per milligram of mitochondrial protein of P. antarcticum were up to eight times higher than state III rates reported in the literature for other notothenioids. The comparatively high respiration rates measured in this study may be explained by our approach, which engaged both complexes I and II under conditions of oxidative phosphorylation. State III rates of independently activated complexes I and II were found to range from 42 to 100 % of rates obtained when both complexes were activated simultaneously in the same species. The remarkable tolerance of P. antarcticum OXPHOS toward warmer temperatures was unexpected for an Antarctic stenotherm and may indicate that thermal sensitivity of their mitochondria is not the driving force behind their stenothermy

Mitochondrial Energetics of Benthic and Pelagic Antarctic Teleosts

Antarctic fauna are highly adapted to the frigid waters of the Southern Ocean. This study describes the in vitro temperature sensitivity of oxygen consumption rates measured in liver mitochondria from the pelagic notothenioid Pleuragramma antarcticum between 5 and 35 C. Oxygen fluxes were measured after the addition of millimolar levels of pyruvate, malate, succinate and glutamate (state II, LEAK) and saturating levels of ADP [state III, oxidative phosphorylation (OXPHOS)]. State III respiration significantly decreased above 18.7 C. A comparison of the oxidative capacities among P. antarcticum and other notothenioids showed significant differences in state III respiration, where benthic species exhibited about 50 % lower rates than P. antarcticum . In addition, state III respiration rates normalized per milligram of mitochondrial protein of P. antarcticum were up to eight times higher than state III rates reported in the literature for other notothenioids. The comparatively high respiration rates measured in this study may be explained by our approach, which engaged both complexes I and II under conditions of oxidative phosphorylation. State III rates of independently activated complexes I and II were found to range from 42 to 100 % of rates obtained when both complexes were activated simultaneously in the same species. The remarkable tolerance of P. antarcticum OXPHOS toward warmer temperatures was unexpected for an Antarctic stenotherm and may indicate that thermal sensitivity of their mitochondria is not the driving force behind their stenothermy

Effects of Osmotic Stress on DNA and Cell Viability in a Desiccation-Sensitive Cell Line

Kc167 is a widely used Drosophila cell line, known to be sensitive to the extreme water loss caused by desiccation. In order to characterize the effects of this desiccation-sensitivity on DNA and cell viability, a series of osmotic stressors of differing concentrations were introduced to the cell line. These cells were then imaged via the Cytation1 cell imaging machine using fluorescence microscopy. Specifically, cells were stained using the DAPI staining solution, a blue fluorescent DNA stain that binds strongly to A-T rich regions within the DNA, forming a fluorescent complex. As DAPI more readily enters the membrane and thereby stains dead cells, instances of apoptosis caused by osmotic stress on cells can be characterized by increasing intensity of fluorescence. Both sucrose and sodium chloride were used to simulate the water loss relevant to that of desiccation. This was done in concentrations of 100mM, 250mM, and 500mM for both sucrose and sodium chloride

Group 3 late embryogenesis abundant proteins from embryos of Artemia franciscana : structural properties and protective abilities during desiccation.

Group 3 late embryogenesis abundant (LEA) proteins are highly hydrophilic, and their expression is associated with desiccation tolerance in both plants and animals. Here we show that two LEA proteins from embryos of Artemia franciscana, AfrLEA2 and AfrLEA3m, are intrinsically disordered in solution but upon desiccation gain secondary structure, as measured by circular dichroism. Trifluoroethanol and sodium dodecyl sulfate are both shown to induce a-helical structure in AfrLEA2 and AfrLEA3m. Bioinformatic predictions of secondary-structure content for both proteins correspond most closely to conformations measured in the dry state. Because some LEA proteins afford protection to desiccation-sensitive proteins during drying and subsequent rehydration, we tested for this capacity in AfrLEA2 and AfrLEA3m. The protective capacities vary, depending on the target enzyme. For the cytoplasmic enzyme lactate dehydrogenase, neither AfrLEA2 nor AfrLEA3m, with or without trehalose present, was able to afford protection better than that provided by bovine serum albumin (BSA) under the same conditions. However, for another cytoplasmic enzyme, phosphofructokinase, both AfrLEA2 and AfrLEA3m in the presence of trehalose were able to afford protection far greater than that provided by BSA with trehalose. Finally, for the mitochondrial enzyme citrate synthase, 400-mg/mL AfrLEA3m without trehalose provided significantly more protection than the same concentration of either AfrLEA2 or BSA

Sonoporation-mediated loading of trehalose in cells for cryopreservation.

Trehalose, a non-reducing disaccharide, is present in many microorganisms and metazoans. In these organisms, trehalose acts as a stress protectant and helps preserve lipid membranes of cells during states of desiccation and freezing. Trehalose is required on both sides of the cell membrane to achieve a significant cryoprotective effect. Specific loading methods for trehalose are required since this sugar is impermeant to mammalian cells. Trehalose loading in mammalian cells has been achieved by fluid-phase endocytosis and genetic modification for the expression of trehalose transporters, however cryoprotective outcomes are unable to compete with established methods of cryopreservation for mammalian cells. Sonoporation was achieved using a microfluidics device modified with an ultrasound emitter in the presence of microbubbles. Ultrasound frequencies emitted by the transducer result in a process called cavitation, which is the rapid expansion and collapse of lipid-coated gas-filled bubbles present in the solution. Cavitation of microbubbles creates small jets of liquid that can create membrane pores that are 150-300 nm in size and quickly reseal through budding and exocytosis allowing for uptake of impermeant compounds, such as trehalose