Latitudinal variation of freeze tolerance in intertidal marine snails of the genus Melampus (Gastropoda: Ellobiidae).

Abstract Low temperatures limit the poleward distribution of many species such that the expansion of geographic range can only be accomplished via evolutionary innovation. We have tested for physiological differences among closely related species to determine whether their poleward latitudinal ranges are limited by tolerance to cold. We measured lower temperature tolerance (LT50) among a group of intertidal pulmonate snails from six congeneric species and nine locales. Differences in tolerance are placed in the context of a molecular phylogeny based on one mitochondrial (cytochrome oxidase subunit I) and two nuclear (histone 3 and a mitochondrial phosphate carrier protein) markers. Temperate species from two separate lineages had significantly lower measures of LT50 than related tropical species. Range differences within the temperate zone, however, were not explained by LT50. These results show that multiple adaptations to cold and freezing may have enabled range expansions out of the tropics in Melampus. However, northern range limits within temperate species are not governed by cold tolerance alone.

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.

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.

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.

Long-term preservation of dried phosphofructokinase by sugars and sugar/zinc mixtures.

We have demonstrated that sugars and suger/zinc mixtures can be used to preserve the activity of dried phosphofructokinase (PFK) during long-term storage over CaSO4. After 9 weeks in the presence of either 200 mM sucrose or 200 mM trehalose little loss of PFK activity was noted, with almost 60% of the original prefreeze-dry activity recovered when samples were rehydrated. Even reducing sugars protected the dried enzyme throughout the entire storage period. Of the sugars tested, 200 mM lactose provided the most stability to PFK; at the end of the dry storage, over 80% of the initial activity was recovered. With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity was recovered at the end of the experiment. With all the sugars tested, the addition of 0.6 mM Zn2+ to sugar/PFK mixtures enhanced the stability of the enzyme, and no long-term adverse effects of the metal ion on enzyme activity were noted.