Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.     

Biotic communities of freshwater marshes and mangroves in relation to saltwater incursions: implications for wetland regulation

An ecosystem-level study was conducted in the Guandu wetlands insubtropical coastal Taiwan to examine how salinity influences the abundance,diversity, and structure of biotic communities. We surveyed eight permanentstudy sites, spanning freshwater marshes, to the gate on the dyke, andmesohaline mangroves representing a gradient of the extent of saltwaterincursions. Analyses of abiotic variables showed that salinity was the primarydetermining factor for discriminating habitat types in the wetlands, butcommunities differed in their sensitivity to salinity. The composition of plantand insect communities was most affected by the salinity gradient, suggestingthe utility of these communities for ecological monitoring of saltwaterincursions. However, spatial changes in communities at higher trophic levels,including macrobenthos, mollusks, fish, and birds, could not be explained simplyby the salinity gradient. Instead, changes in these communities were morerelevant to the composition of other biotic communities. Our results show thatspecies richness and diversity of plant communities were higher in the marshesthan in the mangroves. Nevertheless, insect communities censused in themangroves had higher diversity, despite lower abundance and species richness.Macrobenthos surveyed in the mangroves showed higher biomass and number of taxa.Mollusks and fish were also more abundant at sites near the gate compared to themarsh sites. This suggests that maintaining a tidal flux by means of gateregulation is necessary for conserving the spatial heterogeneity andbiodiversity of coastal wetlands.     

Blue-green algae (cyanobacteria): prospects and perspectives

Summary Photosynthetic, prokaryotic blue-green algae (cyanobacteria) occur in a wide range of natural habitats of diverse ionic composition and as such, represent an important source of biological material for biosolar energy conversion programs using saline water. The gasvacuolate, filamentous Spirulina is grown in seminatural culture in Lake Texcoco, Mexico, as a major source of single-cell protein for animal nutrition. Pilot-scale trials in other areas of the world have also demonstrated the suitability of blue-green algae, including Spirulina, for growth under brackish conditions. The carbohydrate accumulation profiles of blue-green algae differ in isolates from freshwater, marine and hypersaline habitats, with a trend towards sucrose or trehalose accumulation in stenohaline freshwater strains grown in media containing NaCl, while euryhaline and marine forms frequently accumulate glucosylglycerol. Many halotolerant isolates from hypersaline habitats accumulate glycinebetaine in response to osmotic stress. This knowledge may provide scope for future improvement in the N₂ fixation rates of blue-green algae in saline media, using betaine-accumulating N₂-fixing strains in preference to other, saltsensitive isolates.     

Changes in intracellular and extracellular α-amino acids in Gloeothece during N2-fixation and following addition of ammonium

Changes in extracellular and intracellular free amino acids were followed during cyclic phases of N₂-fixation (acetylene reduction) by cultures of the axenic, non-heterocystous cyanobacterium Gloeothece incubated under alternating light and darkness or continuous illumination. Changes in intracellular amino acids were minor, with only arginine (increasing during N₂-fixation) and glutamate (decreasing during fixation) showing significant changes in cells incubated under 12 h light: 12 h dark. The intracellular concentration of glutamine in cultures was always very low and the value of the ratio glutamine: glutamate (GLN:GLU), used as an index of C–N status in eukaryote microbes, was consistently less than 0.05 suggesting that the cells were nitrogen-stressed. On addition of ammonium, there was a transient accumulation of intracellular glutamine, and the ratio GLN:GLU increased rapidly to a value greater than 0.5, typical of unstressed eukaryotes. In contrast to intracellular amino acids, there were significant changes in extracellular amino acids in cultures incubated under alternating light and darkness. Glycine, serine and alanine were released during the dark phase and were taken up again in the light, paralleling the diurnal pattern of nitrogenase activity (high in darkness). It is postulated that this release is usually retained in the mucilage surrounding the cells (but disturbed during even gentle filtration) and that this mucilage may constitute an extracellular vacuole.     

Extra- and intracellular ice formation in mouse oocytes

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.     

Spatial-temporal distribution of Aureoumbra lagunensis (“brown tide”) in Baffin Bay,Texas

Once limited to the Laguna Madre of Texas, blooms of the brown tide organism, Aureoumbra lagunensis, have recently been reported elsewhere. Previous studies have focused on the role of extreme hypersalinity and lack of grazing pressure as facilitators of brown tide blooms. However, development of blooms in systems that are not experiencing extreme hypersalinity, and also that are undergoing eutrophication, suggests that our understanding of A. lagunensis bloom dynamics requires additional refinement. The goal of this study was to quantify the spatial-temporal distribution of, and potential controls upon, A. lagunensis in Baffin Bay, Texas. Five sites were sampled monthly over a three-year period, encompassing nearly two years of drought and hypersaline conditions, followed by a high rainfall, lower salinity period. A. lagunensis abundances were higher during drought in May 2013 – March 2015 compared to the higher rainfall period of April 2015 – April 2016. Abundances typically peaked in summer months, though the seasonal pattern was disrupted in 2015 during the shift from high to low salinity conditions. Persistently high abundances of A. lagunensis were observed in the Laguna Salada tributary of Baffin Bay, which typically has higher dissolved organic nitrogen concentrations and may be less well flushed than other parts of Baffin Bay. Thus this location may serve as a reservoir for A. lagunensis in the system. Overall, A. lagunensis abundance was positively correlated with DOC and salinity, and negatively correlated with ammonium, orthophosphate, and ciliate biovolume. These results suggest a variety of physical, chemical and biological factors affect A. lagunensis population dynamics and stress the need for more research on nutrient-A. lagunensis relationships.     

Temporal and Spatial Habitats of Anadromous Brook Charr in the Laval River and its Estuary

Anadromy in brook charr occurs across the species’ range, but few studies have examined contiguous seasonal movements and habitat utilization of freshwater and estuarine environments. We used acoustic telemetry to track movements of anadromous brook charr for 1 year in the Laval River, Quebec. Fish entered the marine environment in May and June, inhabited shallow (<1.7 m), near-shore areas (<500 m from shore), and rarely ventured beyond the headlands of the bay. They were found where salinities were 1–34 ppt and temperatures 5–18°C. There was a strong tidal periodicity to observed movements with fish running up into bays as they became inundated at high tide and then returning to deep river channels in the bays at low tide. Between late July and early September, the charr returned to freshwater and spawned in the middle and upper sections of the river in October and November. Winter habitats were dispersed over 22 km of the river including two lakes. These detailed observations are an important contribution to our understanding of the evolutionary significance of anadromy in this species. The salinity and temperature co-tolerance provides insights into the species’ post-glacial dispersal, present-day distribution, and potential habitat maps for conservation, restoration, and enhancement programmes.     

Fishes and salinities in the St Lucia estuarine system—a review

The recorded salinity ranges of freshwater, estuarine and marine fish species in Lake St Lucia, a Ramsar and World Heritage Site, are documented. The freshwater group is most diverse and abundant under oligohaline conditions, although the Mozambique tilapia (Oreochromis mossambicus) was common under all salinity regimes. Estuary resident species also favoured oligohaline conditions but, in contrast to the freshwater taxa, were well represented in salinities up to 40 ‰. The marine group was most diverse and abundant within the salinity range 10–40 ‰, but a large number of species could also be found in salinities up to 70 ‰. Very few fish species were able to tolerate salinities between 70 ‰ and 110 ‰, with only O. mossambicus surviving for extended periods in salinities above 110 ‰. All the aquatic macrophytes and most of the zoobenthos within the lake appear to die out within the salinity range of 50–60 ‰, thus creating additional stress to those fish present under such conditions. The food resources least affected by extreme hypersalinity are the microphytobenthos and detritus food chains, with detritivorous fishes being dominant when the lake is in this state. Mass mortalities of fishes in Lake St Lucia have been recorded under both low (<5 ‰) and high salinity (>70 ‰) conditions. The fish kills are often triggered by exceptionally low or high water temperatures which affect the osmoregulatory abilities of these species. Hypersaline conditions and fish mortalities under the most recent closed estuary mouth conditions (2002–2005) are reviewed. If the surface area of St Lucia (35,000 ha) is compared to the total surface area of all South African estuaries (approximately 70,000 ha), then the possibility exists that the loss of the Lake St Lucia nursery area for estuary-associated marine fish species over the past few years may cause significant short-term declines in the future abundance of these taxa on both a local and regional scale.     

Resistance to freshwater exposure in White Sea Littorina spp. II: Acid-base regulation

Parameters of acid-base and energy status were studied by in vivo ³¹P-nuclear magnetic resonance spectroscopy in three White Sea Littorina spp. (L.littorea, L. saxatilis and L. obtusata) during prolonged anaerobiosis in freshwater. Intracellular pH decreased significantly, especially during the early period of anaerobiosis, but later the decrease in intracellular pH slowed down considerably, suggesting a capacity for intracellular pH regulation in all three species. There was a trend for intracellular pH to fall most rapidly in the least freshwater-resistant species, L. obtusata, as compared to the most resistant, L. littorea. Non-bicarbonate, non-phosphate buffer values estimated by the homogenate technique were similar in the three studied species (28–37 mmol pH⁻¹ kg⁻¹ wet weight) and did not change during freshwater exposure. The CaCO₃ buffer value of the foot tissues was considerably higher (171–218 mmol pH⁻¹ kg⁻¹ wet weight) and decreased significantly during freshwater exposure. The contribution of the multiple tissue buffering systems to intracellular pH regulation in Littorina spp. shifts between different stages of freshwater exposure. Initially, the non-bicarbonate, non-phosphate tissue buffering system seems to be of major importance for metabolic proton buffering at intracellular pH between 7.5 and 7.0. During later stages of anaerobiosis and at lower intracellular pH, the CaCO₃ buffer is involved in proton buffering. Decrease in the CaCO₃ buffer value during freshwater exposure was in quantitative agreement with the amount of metabolic protons buffered, thus suggesting that CaCO₃ tissue stores may serve as a major buffering system during prolonged anaerobiosis in Littorina spp. Accepted: 23 December 1999     

Influence of a deep artificial entrance channel on the biological characteristics of the blue swimmer crab Portunus pelagicus in a large microtidal estuary

The opening of a deep and permanent artificial entrance channel into the Peel-Harvey Estuary in southwestern Australia in 1994 provided an excellent opportunity to determine the biological characteristics of the blue swimmer crab Portunus pelagicus in a large microtidal estuary, after tidal exchange with the ocean had increased markedly and salinities recovered more rapidly from their winter minima. P. pelagicus was collected from the original entrance channel, which is located 15 km to the north of the artificial channel, the two large basins (Peel Inlet and Harvey Estuary) and the saline lower reaches of a major tributary at regular intervals in 1995-1998. The resultant information on densities, size and age compositions, growth rates and reproductive biology were compared with those derived from data recorded by Potter et al. [Mar. Biol. 78 (1983) 75] for P. pelagicus in the Peel-Harvey Estuary in 1980 and 1981. This led to the following conclusions regarding the ways in which this portunid has responded to the impacts of the construction of an artificial entrance channel in this estuary. (i) Late 0+ crabs enter earlier than previously the Peel Inlet and both the Harvey Estuary, which now opens directly to the sea, and a tributary river in which salinities likewise rise more rapidly in the spring. (ii) Female crabs become ovigerous earlier, in concert with the earlier restoration of high salinities in the spring. (iii) Ovigerous crabs emigrate to the ocean sooner, presumably in response to the greater stimulus provided for emigration by the marked increase that has occurred in tidal flow. (iv) The growth rate during the first few months of life has increased, possibly as a consequence of a decline in the density of crabs within the estuary during those months. (v) A cohort of small 0+ crabs, not previously found in the estuary and almost certainly derived from population(s) spawning further south, is present for a few months but then disappears from the estuary. Although large crabs do not now appear to grow towards their maximum size as rapidly as previously, this is an artefact brought about by the culling of large crabs through the marked increase that has occurred in fishing pressure during recent years.     

盐胁迫对甜菊细胞内游离Ca2+浓度的影响

钙离子不仅是植物生长发育所需的一种大量元素,而且起了偶联细胞外刺激与胞内反应第二信使的作用,是多种受体激动后信息传递过程的中心环节。近年的研究表明:高温、干旱、触摸、低渗、低温、风、机械刺激、病原菌感染等多种环境胁迫均能引起胞质Ca~(2+)水平的改变。这种变化被认为是植物细胞感受环境胁迫的原初反应之一,它通过启动基因表达和胞内一系列生理生化过程调节细胞对环境改变的适应反应。胞质Ca~(2+)水平的改变有两种来源:胞外Ca~(2+)跨膜内流和胞内钙库如内质网中的Ca~(2+)释放。其中由肌醇磷     

Shifts in the intracellular ATP pools of immobilisedNostoc cells (Cyanobacteria) induced by water stress

Summary Immobilised, desiccated cells ofNostoc commune UTEX 584 have the capacity to increase the size of their extractable intracellular ATP pool upon rewetting. The time taken to recover the pool size depends on the conditions of storage at a particular water potential and the duration of storage. Under the conditions employed, the rewetting of cells induced an increase in ATP pool size at the expense of photophosphorylation or electron transport (oxidative) phosphorylation. The rise in the ATP pool size was instantaneous and was shown to be due to ATP synthesis. This increase did not occur when cells were rewetted in the presence of sodium azide (10 mmol/l), while a partial inhibition was observed with CCCP (carbonyl cyanidem-chlorophenylhydrazone; 2 mol/l). For cells dried at more extreme water potentials, the lag ofc 48 h observed before the ATP pool reached control values is of similar duration to that observed in the recovery of nitrogenase upon rewetting. Chloramphenicol (10 mol/l) stimulated significantly the upshift in the size of the ATP pool ofNostoc cells upon rewetting, yet inhibited completely the rise in nitrogenase activity.     

Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to -50 degrees C at 10 degrees C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as "flashing." Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200 300 microm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is -11.4 degrees C, although a sizeable percentage flash at temperatures much closer to that of the EIF (-3.9 degrees C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to approximately -20 degrees C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to -29 degrees C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128-138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size.     

Halophilic-blue-green algae

The isolation of a halophilic blue-green alga, Aphanothece halophytica, from Great Salt Lake is described. The organism was cultured from waters with salinities up to saturated NaCl (about 30% w/v). It has an optimum salinity for growth of about 16% NaCl, but can grow very slowly even in saturated NaCl. Based on the study of the Great Salt Lake organism, and on a review of the earlier literature, it is concluded that despite recent reports to the contrary, true halophilic blue-green algae do exist.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.