Cold-hardiness of the arctic beetle, Pytho americanus Kirby Coleoptera,Pythidae (Salpingidae)

The arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages. This is the first demonstration that an insect can tolerate freezing in more than one life stage, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold hardening process of low temperature acclimation are increasing glycerol and decreasing glycogen concentrations. Glycerol is the only polyol to be synthesized during acclimation, and it accumulates to a maximum of 8.2 and 12.2% of the fresh body weight in larvae and adults respectively. This coincides with the peak of frost tolerance. In addition to its normally assumed roles in cryoprotection it is suggested that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of ‘bound’ water. Evidence is presented that indicates that glycerol is synthesized mainly from carbohydrate reserves, especially glycogen, but it does not rule out the possibility that a proportion of free glycerol comes from glyceride sources.P. americanus larvae and adults have low supercooling potential and maintain their supercooling points in the region of ?4° to ?8°C. It is hypothesized that these elevated supercooling points are a result of the presence in the haemolymph of nucleating agents which ensure ice formation at high sub-zero temperatures. It is believed that this beetle overwinters in a frozen state within its microhabitat, which is under bark of fallen spruce which is, in turn, covered by an insulating blanket of snow. The advantages of this overwintering strategy are discussed.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Pre-cold acclimation promotes cryoprotectant accumulation and enhances freezing tolerance of Meloidogyne incognita

Strong evidence suggests that cryoprotectant accumulation during pre-cold acclimation protects cells against freezing injuries caused by cellular dehydration. In this study, the concentrations of trehalose and glycerol were measured in Meloidogyne incognita and it was found that both cryoprotectants were significantly accumulated in second-stage juveniles (J2) of M. incognita after acclimation at 4°C. However, compared with non-acclimated samples, only a higher level of trehalose was induced in the egg masses of M. incognita in response to cold treatment. Further characterizations indicated that pre-cold acclimation efficiently accelerated the speed of larvae hatching from egg masses that were subjected to freezing at −1°C. In addition, the survival rate and pathogenicity of M. incognita J2 that had been acclimated prior to freezing were significantly enhanced when compared with non-acclimated J2 individuals. As far as we know, this is the first time that this phenomenon has been reported in M. incognita.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Winter profiles of a natural population

Larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) utilize a freeze-avoidance strategy for winter survival. Cold-hardiness adaptations of an outdoor population of the species were profiled over the 1984–1985 winter. Over the autumn months supercooling points of the larvae dropped from −13.9±2.3°C to −37.8±2.8°C (the lowest winter temperature recorded was −26°C), water content of the larvae decreased from 57.2±1.2 to 24.8±1.6% of fresh weight, and glycerol content of the larvae rose to an average of 2030 μmol/g wet weight or 18.7% of fresh weight. All parameters stabilized over the mid-winter months. Glycerol production was largely accounted for by the loss of stored glycogen while lipid and protein reserves remained nearly constant over the winter months. Supercooling-point depression and glycerol systhesis both appeared to be initiated after the first overnight exposures to subzero temperatures. Highest rates of glycerol production, about 60 μmol g⁻¹ d⁻¹, were achieved with mean daily temperatures of about 0°C and subzero nights. Glycerol content was rapidly cleared in the spring but only 20% of the resulting carbon was restored as glycogen.     

Cold-induced glycerol accumulation by Ostrinia nubilalis larvae is developmentally regulated

Ostrinia nubilalis larvae reared under both nondiapause and diapause-inducing conditions were chilled at 5°C for various periods and their haemolymph glycerol concentrations were measured enzymatically. The ability of fifth (final) instars to accumulate glycerol was dependent upon cold stress but not the diapause state. Furthermore this response was independent of any cold-induced release of cephalic or thoracic hormones. The capacity of O. nubilalis larvae to express cold-induced glycerol accumulation was found to require ecdysis from the fourth to fifth instar. Eggs as well as second, third and fourth instars were completely incompetent. These results indicate that, at the biochemical level, a specific developmental programme or sequence is required for O. nubilalis to demonstrate this response to cold stress.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Laboratory investigations of temperature cues in the regulation of cold hardiness

Laboratory manipulations of ambient temperature were used to investigate the role of temperature in triggering or modulating cold-hardiness adaptations, supercooling-point depression and cryoprotectant accumulation, in larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens), a freeze-intolerant species. Low temperature strongly facilitated cryoprotectant synthesis; larvae subjected to a 1°C per day decrease in temperature showed a major increase in the rate of glycerol synthesis when temperature fell below 5°C with highest rates of synthesis, greater than 90 μmol g⁻¹ d⁻¹, at temperatures between 0 and −10°C. Conversely, abrupt rewarming of larvae from −18 to 23°C in mid-November stimulated a rapid loss of glycerol (from a starting level of 1763 ± 278 μmol/g wet weight) with a half time of only 1.5 days. Supercooling-point depression was not keyed to ambient temperature but appeared to be an endogenous event occurring over the same time interval in laboratory animals held at warm or cold temperatures, as well as in outdoor animals. Rewarming of cold-adapted larvae in November resulted in only a small rise in supercooling point (and did not break diapause) but rewarming in February resulted in a 19°C increase in supercooling point in 4 days, followed rapidly by pupation.     

Glycerol permeability of rye leaf protoplasts as affected by temperature and electroporation

The permeability of rye leaf protoplasts to glycerol was determined using 1,3-¹⁴C glycerol and liquid scintillation spectrometry. Estimates were 1.0×10⁻⁸ m s⁻¹ at 0°C and 4.1×10⁻⁸ m s⁻¹ at 22 and 31°C. The activation energy for glycerol permeability was 32.8 kJ/mol. The effect of electroporation on glycerol uptake was also explored. Treatments were performed with a field strength of 100 V/cm and an exponential decay constant of 5.8 ms. At 22 °C, electroporation affected the rate and extent of glycerol permeation, causing an increase in the intercept of the glycerol uptake curve and a decrease in the slope. Electroporation had no significant effect on glycerol uptake when performed at 0°C, when the cells were electroporated at 0°C then warmed to 31 °C, or when the cells were electroporated at 22 °C then cooled to 0°C. The results at 22°C were consistent with an influx of glycerol during electroporation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of cold stress on juvenile Piaractus mesopotamicus and the mitigation by β-carotene

This study investigated the effects of cold stress on morphometrical and hematological biomarkers, energy metabolism, and oxidative stress in different tissues of P. mesopotamicus, and the protective role of β-carotene. Fish were fed with a control diet (CD) and the same diet supplemented with 105 mg/kg β-carotene (BD) for 60 days. After the feeding trial, fish fed CD or BD diets were exposed to control (24 °C) and low temperature (14 °C) for 24 h. Fish (CD and BD) exposed to thermal stress showed lower hepatosomatic index. The hemoglobin increased only in CD-fed fish exposed to 14 °C. Increased glycemia, plasmatic protein depletion, and decreased hepatic glycogen were observed in fish fed the CD, while only the lipid levels in liver were augmented in BD-fed fish exposed at 14 °C. Regarding the oxidative stress, increased antioxidant enzymes activity and lipid peroxidation were observed in CD-fed fish exposed to cold. The two-way ANOVA showed an interaction between dietary treatment and temperature for glucose and oxidative stress biomarkers, with the highest values recorded in 14 °C-exposed fish fed with the CD. Our study demonstrated that cold stress had the greatest impact on fish oxidative status, and β-carotene reduces harmful effects induced by cold in P. mesopotamicus.     

Gluconeogenic pathway does not display metabolic cold adaptation in liver of Antarctic notothenioid fish

Antarctic notothenioid fish display specializations related to cope with their chronically cold environment, such as high triacylglycerol (TAG) content in tissues. The metabolic fate of glycerol, a product of TAG mobilization, has not been studied in Antarctic fish. To assess the importance of glycerol as a substrate for gluconeogenesis and to determine whether this pathway is metabolically cold adapted (MCA), key hepatic enzyme activities were measured in Antarctic (Notothenia coriiceps, Gobionotothen gibberifrons, and Chionodraco rastrospinosus) and non-Antarctic (Dissostichus eleginoides, Patagonotothen ramsayi, and Eleginops maclovinus) notothenioid fish. Fructose 1,6-biphosphatase (FBP), phosphoenolpyruvate carboxykinase (PEPCK), and glycerol kinase (GK) activities were similar in both groups at common temperatures (1, 6, 11, or 21 °C). In particular, thermal sensitivity for the reactions catalyzed by FBP and PEPCK was analogous between Antarctic and non-Antarctic species, reflected by similar values for Arrhenius energy of activation (E _a) and Q₁₀. Additionally, hepatic glycerol, glucose, and glycogen contents together with plasma glycerol and glucose concentrations were similar for all of the species studied. Our results do not support the concept of MCA in hepatic gluconeogenesis and may indicate that the use of glycerol as a precursor for glucose synthesis by this pathway is of low physiological importance in Antarctic fish.     

Permeability of intact and dechorionated zebra fish embryos to glycerol and dimethyl sulfoxide

Intact developing embryos of the zebra fish Brachydanio rerio were exposed to [¹⁴C]DMSO and [³H]glycerol (1 M in Fish Ringer) to assess the degree of permeation of these cryoprotectants. Glycerol enters the embryo more easily, although reaching only about 8% of the expected equilibrium level after 2 hr at room temperature; DMSO reaches only about 2.5% of this level. In an attempt to identify the barrier to permeation, dechorionated embryos were similarly exposed to isotopic DMSO. Permeation increased severalfold, indicating that the chorion retards the free exchange of solute. Embryos are unaffected by exposure to 1 M DMSO in Fish Ringer at 23 °C for up to 1 hr. The number of embryos hatching after 1-hr exposure to DMSO at varying concentrations was significantly reduced at 1.5 and 2 M. Embryos exposed to 1 M glycerol for 1 hr at 23 °C showed disruption of periblast cells and separation of the blastoderm; it was impossible to remove glycerol either by abrupt or very slow dilution.     

Protection of photosynthesis and antioxidative system by 24-epibrassinolide in Solanum melongena under cold stress

This study was carried out to understand the mechanism of protection of plants under cold stress by exogenous 24-epibrassinolide (EBR). The eggplant (Solanum melongena L.) seedlings were pretreated with five concentrations of EBR (0, 0.05, 0.1, 0.2 and 0.4 °M) and then exposed to day/night temperatures of 10/5 °C for 8 d. The results show that EBR, especially 0.1 °M EBR, dramatically alleviated growth suppression and a decrease in chlorophyll content and photosynthetic rate caused by the cold stress. In addition, EBR also decreased malondialdehyde content and O₂ ^·? production rate induced by the cold stress, and increased the activities of superoxide dismutase, guaiacol peroxidase, catalase, and ascorbate peroxidase, and proline content. The results of the present study suggest that exogenous EBR could improve cold tolerance of eggplant by regulating photosynthesis and antioxidative systems.     

Comparison of the cold hardiness of two larval Lepidoptera (Noctuidae)

Abstract Diapause larvae of the European corn borer (Ostrinia nubilalis (Hubn.)) and the related Mediterranean noctuid Sesamia cretica Led. possess sufficient supercooling ability to avoid freezing over their normal environmental temperature ranges. In progressive chilling experiments (10 days acclimation at each 5° step in the temperature range from 15 to ?5°C), mean supercooling points (measured at a cooling rate of 0.1°C min^?1) were lowered from ?20.4°C at 15°C to ?24.0°C at 5°C (lower lethal temperatures: c.?28°C) in O.nubilalis, compared with ?15.0 to ?17.2°C (lower lethal temperatures: ?15 to ?17°C respectively) in S.cretica. Concentrations of glycerol and trehalose determined by gas chromatography of whole body extracts were consistently higher in the former than in the latter species at both 15 and 5°C, and may be responsible for the deeper supercooling in O.nubilalis larvae. Acclimation to 5°C increased glycerol levels in O. nubilalis extracts compared with 15°C, and this was enhanced in larvae exposed for a further 10 days at each of 0 and ?5°C (glycerol being 438μmol ml^?1 body water). Haemolymph glycerol concentrations showed a similar pattern to whole body extracts in this species. Fat body glycogen was reduced during low temperature acclimation in both species. Body water contents did not change with acclimation in O.nubilalis, whilst S.cretica, containing significantly more water, lost c.7% during acclimation from 15 to 5°C. Haemolymph osmolalities increased during acclimation, especially in Ostrinia larvae, probably as a result of the accumulation of cryoprotectants. The majority of O.nubilalis larvae survived freezing under the conditions of the cooling experiments, whilst larvae of S.cretica did not, thereby confirming an element of freezing tolerance in the former.     

The cerulenin-induced formation of 1-acyl-lysophosphatidyl glycerol in Bacillus megaterium.

When a culture of $\text{Bacillus megaterium}$ ATCC 14581, growing at 20° and treated with the fatty acid synthesis inhibitor, cerulenin, was incubated with [U-¹⁴C]palmitate, 50% of the incorporated label was found in 1-palmitoyl-lysophosphatidyl glycerol within 5 min. Most of the remaining ¹⁴C appeared in free fatty acid and phosphatidyl glycerol. By 45 min almost all of the lyso compound had disappeared and 80% of the incorporated label was found in phosphatidyl glycerol. At 20°, in the absence of cerulenin or at 35° in either its presence or absence, no labeled lysophosphatidyl glycerol could be found at any time after [U-¹⁴C]palmitate addition. The major radioactive lipid, in these cases, was always phosphatidyl glycerol. At 20°, the palmitate of phosphatidyl glycerol but not of lysophosphatidyl glycerol was readily desaturated.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.     

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

Alleviation of cold stress in inoculated wheat (<Emphasis Type="Italic">Triticum aestivum L</Emphasis>.) seedlings with psychrotolerant Pseudomonads from NW Himalayas

Twelve psychrotolerant Pseudomonad strains were selected on the basis of various plant growth-promoting (PGP) activities at cold temperature (4°C). The effect of inoculation with Pseudomonad strains on cold alleviation and growth of wheat seedling at cold temperature (8°C) was investigated under greenhouse condition. Inoculation with Pseudomonad strains significantly enhanced root/shoot biomass and nutrients uptake as compared to non-bacterized control at 60 days of plant growth. Bacterization significantly improved the level of cellular metabolites like chlorophyll, anthocyanin, free proline, total phenolics, starch content, physiologically available iron, proteins, and amino acids that are sign of alleviation of cold stress in wheat plants. Increased relative water content, reduced membrane injury (electrolyte leakage), and Na⁺/K⁺ ratio were also recorded in bacterized wheat plants. Electrolyte leakage and Na⁺/K⁺ were found inversely proportional to plant growth at cold temperature. Statistical analysis of twenty-three measured parameters revealed that uninoculated control was under cold stress while eight bacterial strains were positively alleviating cold stress in wheat plants. Thus, the psychrotrophic Pseudomonad strains could effectively provide a promising solution to overcome cold stress, which is major factor hindering wheat productivity under cold climatic condition.