Stephen H. Loomis

Mechanisms of interaction of amino acids with phospholipid bilayers during freezing

In this study we compare the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20 degrees C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 mM NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains of less than three carbons long, e.g. glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. We suggest that NaCl selectively increases the solubility of such amino acids, allowing them to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for cryoprotection by these molecules. We present evidence that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup.

Purification and partial characterization of an ice nucleator protein from the intertidal gastropod, Melampus bidentatus

An ice nucleator protein has been purified from the intertidal gastropod Melampus bidentatus by a combination of gel filtration and ion exchange chromatography. Partial characterization of this ice nucleator protein indicates that in the native form, it exists as an aggregate with a molecular weight in excess of 600 kDa. SDS-PAGE analysis indicates that this aggregate is composed of two low molecular weight protein species of approximately 16.6 and 17.4 kDa. Biochemical analysis indicates that it is neither a lipoprotein nor a glycoprotein. The amino acid composition indicates that it contains a high percentage of polar amino acids with Asx and Glx representing over 20 mol% of the protein. The characteristics of this molluskan ice nucleator protein are compared with those of purified ice nucleator proteins obtained from bacteria and various species of insects.

Isolation and identification of an ice-nucleating bacterium from the gills of the intertidal bivalve mollusc Geukensia demissa.

In the fall, freeze tolerant intertidal invertebrates usually produce ice-nucleating proteins that are secreted into the hemolymph. These proteins help protect against freeze damage by insuring that ice formation is limited to extracellular spaces. Geukensia demissa, a freeze tolerant, salt marsh bivalve mollusc was examined for the presence of ice nucleating proteins. The ice-nucleating temperature (INT) of the hemolymph was not significantly different from artificial seawater of the same salinity indicating the lack of an ice nucleating protein in the hemolymph. The palial fluid did have an elevated INT, indicating the presence of an ice nucleator. The INT of the palial fluid was significantly reduced by boiling and filtration through a 0.45-&mgr;m filter. High INT was also observed in the seawater associated with the bivalves, and was demonstrated in water samples collected from salt marshes but not sand and pebble beaches. Moreover, the INT of water samples collected from a salt marsh decreased in the summer. All of these data suggest that the ice-nucleating agents in the hemolymph and the seawater are ice-nucleating bacteria. One species of ice-nucleating bacteria, Pseudomonas fulva was isolated from the gills of Geukensia. These bacteria could perform the same function as hemolymph ice-nucleating proteins by limiting ice formation to extracellular compartments.

Metabolism of Gemmules From the Freshwater Sponge Eunapius fragilis During Diapause and Post-Diapause States

Post-diapause gemmules of the freshwater sponge Eunapius fragilis remained quiescent when maintained at 5°C. Germination occurred within 48 to 72 h following warming to 20°-23°C, culminating with the emergence of a new sponge from the collagenous capsule. Both heat dissipation and oxygen consumption climbed steadily during germination and eventually reached 600% of the starting values. By comparison, energy flow was much lower over the same period of time in diapausing gemmules, clearly demonstrating metabolic depression during diapause. The calorimetric:respirometric (CR) ratio increased significantly from -354 kJ/mol O2 to -541 kJ/mol O2 between hours 3.5 and 56.5 of germination, with an average value across this period of about -495 kJ/mol O2. The low CR ratio at hour 12.5 (-374 +/- 21; +/- 1 SE, n = 3) was statistically below the oxycaloric equivalent, which suggests that gemmules may have experienced hypoxia during the more than 3 months of storage at 5°C prior to experiments. The increase in metabolism during germination could be blocked by perfusing the gemmules with nitrogen-saturated medium (nominally oxygen free). Developing gemmules were able to survive oxygen limitation for several hours at least; during that time energy flow was depressed to 6% of normoxic values. During germination, the range of values was 3.5 to 4.0 nmol/mg protein for ATP, 0.2 to 0.4 nmol/mg protein for ADP, and 0.5 to 0.8 nmol/mg protein for AMP. Because ATP was high even before gemmules were warmed to room temperature, it is unlikely that levels were severely compromised during the diapause condition.

Diapause and Estivation in Sponges

Sponges can be found in fresh or saltwater habitats. As part of their life cycle, many sponges produce gemmules as a means of surviving environmental challenge. In most sponges, the gemmules contain cells that are initially in a state of metabolic arrest that is controlled by endogenous factors. This state is known as diapause. Following a period of exposure to unfavorable conditions, the cells in the gemmule transit from diapause into a state known as quiescence in which metabolic depression is controlled by environmental factors. When favorable conditions return, the gemmules germinate and produce a new sponge. Production of gemmules is triggered by environmental factors such as decreased temperature or desiccation and involves cell aggregation of thesocytes and the laying down of the gemmule coat. Thesocytes contain yolk platelets as an energy store and high concentrations of polyols that maintain high osmotic concentration in the cells of the gemmules. The high osmotic concentration maintains metabolic depression and turns off cell division. It is the inability to reduce the osmotic concentration that maintains the gemmules in diapause. Transition to quiescence requires the ability of the cells in the gemmules to convert the polyols to glycogen, and thus reduce the osmotic concentration. At this stage, the cells are able to reduce osmotic concentration but do not until favorable conditions return. Early in the germination process, the polyols are converted to glycogen, reducing the osmotic pressure and releasing the inhibition of cell division and metabolic rate. Both cell division and metabolic rate increase eventually leading to germination of the gemmules and production of a new sponge.

Carbohydrate Mobilization During Germination of Post-Diapausing Gemmules of the Freshwater Sponge Eunapius fragilis

Post-diapausing gemmules of the freshwater sponge Eunapius fragilis were found to contain sorbitol and glycogen as their primary carbohydrates. The sorbitol probably acts to increase the tolerance of the gemmules to freezing and desiccation. During germination, average sorbitol levels--measured as micromoles of sorbitol per gram of fresh weight of gemmule tissue (μmol/gfw)--declined from a control value of 36 μmol/gfw to about 4 μmol/gfw. Concomitantly, average glycogen levels increased from a control value of 29 μmol/gfw to a steady-state level of 62 μmol/gfw. It is probable that glycogen is being synthesized at the expense of sorbitol. The breakdown of sorbitol was associated with an increase in the activity of sorbitol dehydrogenase from undetectable levels in dormant gemmules to a maximum of 0.2 μmol/ min · mg protein after 30 h of exposure to 20°C. Aldose reductase activity remained constant throughout germination. These data support the hypothesis that the decrease in sorbitol levels is the result of an increase in the rate of catabolism by sorbitol dehydrogenase. The total activity of glycogen synthase did not change during germination; however, the activity of glucose-6-phosphate-dependent glycogen synthase was about 18 times greater than the activity of glucose-6-phosphate-independent glycogen synthase. Total glycogen phosphorylase activity increased from about 1.6 nmol/min.mg protein to 3.6 nmol/min.mg protein during germination. At the same time, however, the percentage of glycogen phosphorylase a decreased from almost 100% to about 84%. This decrease would attenuate the apparent increase in activity. cAMP levels remained constant throughout germination. The observed changes in the level of glycogen in the gemmules are not simply due to changes in the activity of either glycogen phosphorylase or glycogen synthase.

Ice formation and freezing damage in the foot muscle of the intertidal snail Melampus bidentatus

Abstract Freeze injury to the foot muscle of the intertidal pulmonate gastropod, Melampus bidentatus was examined by light microscopy using a technique which allowed fixation of the tissues at subzero temperatures. Exposure of snails to −5 or −10 °C (above the lower lethal temperature) resulted in no apparent damage to the foot muscle. Exposure of the snails to −13 °C (the lower lethal temperature), however, resulted in sloughing of the epithelium and possible loss of membrane material. Ice was present in the extracellular spaces of foot muscle which was fixed at −10 and −13 °C but not in tissues exposed to −5 °C. The percentage of the area occupied by the cells in muscle tissues decreased significantly when ice was present and increased to control levels upon warming in all cases except for the tissue exposed to −13 °C.

The Effects of Elevated Osmotic Concentration on Control of Germination in the Gemmules of Freshwater Sponges Eunapius fragilis and Anheteromeyania ryderi

Freshwater sponges produce gemmules during the fall as an adaptation to survive cold winters. Most gemmules are produced in a state of diapause and must undergo a vernalization period before diapause is broken and they enter the quiescence state. Quiescent gemmules will germinate if placed at room temperature. We examined the mechanism of germination in two species of freshwater sponges, Eunapius fragilis and Anheteromeyania ryderi. Germination, cell division, and oxygen consumption are all inhibited when the osmotic concentration of the gemmules of either species is maintained at or above 50 mOsm by placing them in a solution of impermeable osmolytes. The internal osmotic concentration of cells of quiescent gemmules is maintained above 100 mOsm by the presence of sorbitol (in E. fragilis) and myoinositol (in A. ryderi). During the early stages of germination, levels of sorbitol and myoinositol decline to less than 50 mM by 20 h after the initiation of germination. The onset of cell division and beginning of germination correlate with the drop in osmolyte levels below 50 mOsm. Thus, an early trigger initiating germination is most likely the catabolism of sorbitol or myoinositol leading to a drop in the osmotic concentration of the cells.

Possession and Loss of Cold Tolerance by Sponge Gemmules: A Comparative Study

Gemmules of the freshwater sponges Eunapius fragilis, Ephydatia muelleri, and Spongilla lacustris from southern New England differ in their resistance to exposure to extremely low temperatures. Eunapius fragilis appears to be most tolerant, with greater than 80% of the gemmules surviving exposure to -70? C for one hour. Ephydatia muelleri is slightly less tolerant, and Spongilla lacustris is the least tolerant with no gemmules surviving exposure to temperatures lower than 10? C. Gemmules of both Eunapius fragilis and Ephydatia muelleri lose cold tolerance with storage at 3? to 5? C for more than a few months, most probably due to the loss of cryoprotectants such as sorbitol. Additional key words: Porifera, Eunapius fragilis, Ephydatia muelleri, Spongilla lacustris Most freshwater sponges produce dormant structures called gemmules, each consisting of a dense mass of nutrient-laden cells enclosed by a collagenous capsule that often contains spicules. Some species possess compound gemmules in which each cellular mass is surrounded by its own inner capsule layer but a number of cellular masses share common outer layers. Gemmules may be quiescent, dormancy being imposed by unfavorable environmental conditions, and/ or they may exhibit diapause, in which the dormant state is maintained endogenously (Rasmont 1962; Simpson & Fell 1974; Simpson 1984; Fell 1993). When appropriately tested, the gemmules of freshwater sponges from temperate regions have usually been found to survive exposure to subfreezing temperatures for extended periods of time (Fell 1993). Hydrated gemmules of Ephydatia muelleri in Quebec, Canada survived exposure to 80? C for up to 63 days (Barbeau et al. 1989). Similarly, hydrated gemmules of Eunapius fragilis in Connecticut, USA exhibited more than 80% survival following exposure to -72? C for 30 days (Boutselis et al. 1990); and green gemmules of Spongilla lacustris in Massachusetts, USA showed -80% survival after being frozen in pond water at -20? C for up to 30 days (Fell & Levasseur 1991). Time spent at low temperature has little effect on gemmule survival. However, in some cases, the rate of hatching at 20? C following exposure of gemmules to very low temperatures is markedly slowed compared with that of a Author with whom to correspond. control gemmules which had been maintained at 5? C (Barbeau et al. 1989; Fell & Levasseur 1991). Important factors influencing the cold tolerance of hydrated gemmules of Eunapius fragilis are the rates of cooling and warming. These rates, in turn, affect the rates of ice formation and thawing within the gemmules. Cooling at a rate of 0.1? or 0.5? C/min. and subsequent warming at a rate of 10? C/min. provide optimal conditions for gemmule survival. Under these conditions there was little difference in the survival of gemmules of Eunapius fragilis exposed to -30? -70?, or 100? C (Boutselis et al. 1990). If sponge gemmules are exposed to air during the winter, they may be dehydrated when subjected to subfreezing temperatures (Fell & Bazer 1990). Since little or no ice formation occurs within gemmules under these conditions, it would be expected that cold tolerance should be unaffected by cooling and warming rates. This was found to be the case for dehydrated gemmules of Eunapius fragilis (blotted gemmules were kept in air at 5? C for 24 h before they were tested in air, Boutselis & Fell, unpubl. data). In most cases, the results of earlier studies on the cold tolerances of sponge gemmules cannot be compared directly because each study dealt with a single species and the various studies used different methods. Here we compare the cold tolerances of gemmules of three freshwater sponge species gathered from habitats in southern New England, stored under the same conditions, and then simultaneously exposed to low-temperature conditions. We examined tolerances of the gemmules to different low temperatures over a period This content downloaded from 157.55.39.153 on Mon, 19 Sep 2016 04:42:00 UTC All use subject to http://about.jstor.org/terms Ungemach, Souza, Fell, & Loomis of months in order to ascertain whether changes in cold tolerance occur during maintenance at 4-5? C. Although these sponge gemmules would not be exposed to temperatures as low as -30? or -70? C under natural conditions, we tested the survival of gemmules at these temperatures because we wanted to determine how cold tolerant the gemmules of the different species are. Furthermore, since the amount of extracellular ice formed increases with decreasing temperature, the degree of cold tolerance implies certain physiological conditions within the gemmules.

The use of free amino acids for osmotic compensation by the euryhaline sponge Microciona prolifera (Ellis & Sollander)

Microciona prolifera (Ellis and Sollander) is a euryhaline sponge able to tolerate extended periods of exposure to salinities ranging from 10 to 40‰ and brief exposure to 5 or 45‰ seawater. After a 7-day adjustment period in 30‰ seawater at 20 °C, explants of this sponge were transferred to higher or lower salinities at a rate of 5‰ every 3 days. The explants were acclimated to these salinities (40‰, 20‰ and 10‰) for 2 weeks. Control explants were kept at 30‰. Explants kept at 10‰ underwent a reversible tissue regression characterized by cessation of normal pumping activity and loss of functional internal structure, a state that may enable the organism to tolerate long-term exposure to salinity stress by reducing the amount of surface area in contact with adverse conditions. Exposure to 40‰ resulted in a reduction in pumping activity, but the explants did not undergo extensive tissue regression. Acclimation to lower salinities resulted in a reduction in the intracellular concentration of ninhydrin positive substances, primarily the free amino acids glycine and phenylalanine. As salinity was lowered from 30 to 20‰, the concentrations of glycine and phenylalanine were reduced by 38 and 78%, respectively. The concentrations of these osmolytes were reduced 93 and 91%, respectively, when salinity was lowered from 30 to 10‰. While the concentration of ninhydrin positive substances increased with acclimation to a higher salinity, the calculated total free amino acid concentration remained unchanged.