Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Survival of rapidly fluctuating natural low winter temperatures by High Arctic soil invertebrates

The extreme polar environment creates challenges for its resident invertebrate communities and the stress tolerance of some of these animals has been examined over many years. However, although it is well appreciated that standard air temperature records often fail to describe accurately conditions experienced at microhabitat level, few studies have explicitly set out to link field conditions experienced by natural multispecies communities with the more detailed laboratory ecophysiological studies of a small number of ‘representative’ species. This is particularly the case during winter, when snow cover may insulate terrestrial habitats from extreme air temperature fluctuations. Further, climate projections suggest large changes in precipitation will occur in the polar regions, with the greatest changes expected during the winter period and, hence, implications for the insulation of overwintering microhabitats. To assess survival of natural High Arctic soil invertebrate communities contained in soil and vegetation cores to natural winter temperature variations, the overwintering temperatures they experienced were manipulated by deploying cores in locations with varying snow accumulation: No Snow, Shallow Snow (30 cm) and Deep Snow (120 cm). Air temperatures during the winter period fluctuated frequently between +3 and −24 °C, and the No Snow soil temperatures reflected this variation closely, with the extreme minimum being slightly lower. Under 30 cm of snow, soil temperatures varied less and did not decrease below −12 °C. Those under deep snow were even more stable and did not decline below −2 °C. Despite these striking differences in winter thermal regimes, there were no clear differences in survival of the invertebrate fauna between treatments, including oribatid, prostigmatid and mesostigmatid mites, Araneae, Collembola, Nematocera larvae or Coleoptera. This indicates widespread tolerance, previously undocumented for the Araneae, Nematocera or Coleoptera, of both direct exposure to at least −24 °C and the rapid and large temperature fluctuations. These results suggest that the studied polar soil invertebrate community may be robust to at least one important predicted consequence of projected climate change.     

Individualising the exposure of −110 °C whole body cryotherapy: The effects of sex and body composition

The purpose of this study was to investigate the effects of whole body cryotherapy (WBC) on a range of thermoregulatory measures. We also sought to examine the influence of sex and body composition. A convenience sample of 18 healthy participants (10 males and 8 females) (27±6 yr) volunteered for this study. Temperature (core, tympanic, skin and mean body), heart rate, blood pressure, and thermal comfort and sensation were recorded pre- and post- (immediately and every 5 min until 35 min post) exposure to a single bout of WBC (30 s at −60 °C, 150 s at 110 °C). Anthropometric data (height, weight, body surface area, body mass index, fat mass and fat free mass) were also recorded. No significant differences in temperature (core, tympanic, skin and mean body), heart rate, blood pressure, or thermal comfort / sensation were observed between male and females at baseline. Immediately post WBC mean body (male:31.9±0.8 °C; female:31.0±0.9 °C; ∆ mean body temperature:0.9±0.1 °C; P≤0.05, d=0.64) and mean skin (male:22.1±2.2 °C; female:19.6±2.8 °C; ∆ mean skin temperature:−2.5±0.6 °C; d=0.99, P≤0.05) temperature was significantly different between sexes. Sex differences were also observed in regional skin temperature (male thigh, 20.8±1.1 °C; female thigh, 16.7±1.1 °C, ∆ mean thigh skin temperature:−4.1 °C; d=3.72; male calf, 20.5±1.1 °C; female calf, 18.2±1 °C, ∆ mean calf skin temperature:−2.3±0.1 °C; d=3.61; male arm, 21.7±1 °C; female arm, 19±0.4 °C, ∆ mean arm skin temperature: −2.7±0.3 °C; d=3.54; P≤0.05). Mean arterial pressure was significantly different over time (P≤0.001) and between sexes (male 0 mins:94±10 mmHg; female 0 mins:85±7 mmHg; male 35 mins:88±7 mmHg; female 35 mins:80±6 mmHg; P≤0.05). Combined data set indicated a strong negative relationship between skin temperature and body fat percentage 35 min’ post WBC (r=−0.749, P≤0.001) and for core temperature and body mass index in males only (r=0.726, P≤0.05) immediately after WBC. There were no significant differences between sexes in any other variables (heart rate, tympanic and perceptual variables). We observed sex differences in mean skin and mean body temperature following exposure to whole body cryotherapy. In an attempt to optimise treatment, these differences should be taken into account if whole body cryotherapy is prescribed.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Validation of a new whole-body cryotherapy chamber based on forced convection

Whole-body cryotherapy (WBC) and partial-body cryotherapy (PBC) are two methods of cold exposure (from −110 to −195 °C according to the manufacturers). However, temperature measurement in the cold chamber during a PBC exposure revealed temperatures ranging from −25 to −50 °C next to the skin of the subjects (using isolating layer placed between the sensor and the skin). This discrepancy is due to the human body heat transfer. Moreover, on the surface of the body, an air layer called the boundary layer is created during the exposure and limits heat transfer from the body to the cabin air. Incorporating forced convection in a chamber with a participant inside could reduce this boundary layer. The aim of this study was to explore the use of a new WBC technology based on forced convection (frontal unilateral wind) through the measurement of skin temperature. Fifteen individuals performed a 3-min WBC exposure at −40 °C with an average wind speed of 2.3 m s⁻¹. The subjects wore a headband, a surgical mask, underwear, gloves and slippers. The skin temperature of the participants was measured with a thermal camera just before exposure, just after exposure and at 1, 3, 5, 10, 15 and 20 min after exposure. Mean skin temperature significantly dropped by 11 °C just after exposure (p<0.001) and then significantly increased during the 20-min post exposure period (p<0.001). No critically low skin temperature was observed at the end of the cold exposure. This decrease was greater than the mean decreases in all the cryosauna devices with reported exposures between −140 °C and −160 °C and those in two other WBC devices with reported exposures between −60 °C and −110 °C. The use of this new technology provides the ability to reach decreases in skin temperature similar to other technologies. The new chamber is suitable and relevant for use as a WBC device.     

Explicit formula of finite difference method to estimate human peripheral tissue temperatures during exposure to severe cold stress

During cold exposure, peripheral tissues undergo vasoconstriction to minimize heat loss to preserve the maintenance of a normal core temperature. However, vasoconstricted tissues exposed to cold temperatures are susceptible to freezing and frostbite-related tissue damage. Therefore, it is imperative to establish a mathematical model for the estimation of tissue necrosis due to cold stress. To this end, an explicit formula of finite difference method has been used to obtain the solution of Pennes' bio-heat equation with appropriate boundary conditions to estimate the temperature profiles of dermal and subdermal layers when exposed to severe cold temperatures. The discrete values of nodal temperature were calculated at the interfaces of skin and subcutaneous tissues with respect to the atmospheric temperatures of 25 °C, 20 °C, 15 °C, 5 °C, −5 °C and −10 °C. The results obtained were used to identify the scenarios under which various degrees of frostbite occur on the surface of skin as well as the dermal and subdermal areas. The explicit formula of finite difference method proposed in this model provides more accurate predictions as compared to other numerical methods. This model of predicting tissue temperatures provides researchers with a more accurate prediction of peripheral tissue temperature and, hence, the susceptibility to frostbite during severe cold exposure.     

The influence of cryopreservation and quick-freezing on the mechanical properties of tendons

In order to maintain their native properties, cryopreserved tendons are usually used in biomechanical research and in transplantation of allogenic tendon grafts. The use of different study protocols leads to controversy in literature and thus complicates the evaluation of the current literature. The aim of this study consisted in examining the influence of different freezing and thawing temperatures on the mechanical properties of tendons. 60 porcine tendons were frozen at either −80 °C or −20 °C for 7 days and thawed at room or body temperature for 240 or 30 min, respectively. A subgroup of ten tendons was quick-frozen with liquid nitrogen (−196 °C) for 2 s before cryopreservation. Biomechanical testing was performed with a material testing machine and included creep, cyclic and load-to-failure tests. The results showed that freezing leads to a reduced creep strain after constant loading and to an increased secant modulus. Freezing temperature of −80 °C increased the secant modulus and decreased the strain at maximum stress, whereas thawing at room temperature reduced the maximum stress, the strain at initial tendon failure and the Young’s Modulus. Quick-freezing led to increased creep strain after constant loading, increased strain at initial failure in the load-to-failure test, and decreased strain at maximum stress. When cryopreserving, tendons for scientific or medical reasons, freezing temperature of −20 °C and thawing temperature of 37.5 °C are recommended to maintain the native properties of tendons. A treatment with liquid nitrogen in the sterilization process of tendon allografts is inadvisable because it alters the tendon properties negatively.     

Effect of storage conditions of blood on radiation-induced chromosomal aberrations and apoptosis in human lymphocytes

To evaluate the effect of storage conditions of blood on the direct relationship between radiation-induced chromosome aberrations and apoptosis in human peripheral blood lymphocytes, whole blood was irradiated with 3 Gy X-rays. Directly after irradiation, a sample of blood was analyzed for chromosome damage and proliferation index, after phytohaemagglutinin stimulation and incubation at 37 °C for 56 h. Blood samples were stored for 48 h at 4 and 20 °C with or without phytohaemagglutinin and analyzed for cell viability and apoptosis at 0, 24 and 48 h storage time. After 48 h of storage, unstimulated cultures were stimulated to proliferate. These samples and cultures stimulated immediately before storage were incubated at 37 °C for 56 h and analyzed for chromosome damage and proliferation index. Metaphases were examined for the presence of dicentrics, excess acentrics, and rings. Storage at 20 °C without phytohaemagglutinin for 48 h increases significantly the yield of apoptosis and decreases significantly the yield of dicentrics. During 48 h of storage time the presence of phytohaemagglutinin and the temperature of 4 °C protected the irradiated lymphocytes from apoptosis allowing accurate estimation of the real yield of radiation-induced chromosome damage. Therefore these blood-storage conditions enable analysis in metaphase and may offer some advantages for biodosimetry of absorbed radiation dose.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.     

Effects of sample handling,processing, storage,and hemolysis on measurements of key energy metabolites in ovine blood

Four experiments were conducted using mature Suffolk ewes to evaluate the effects of blood sample handling, processing and storage on measurements of the energy metabolites, β-hydroxybutyrate, total cholesterol, glucose, l-lactate, nonesterified fatty acid (NEFA), and triacylglycerol. In experiment 1 the effects of anticoagulants on metabolite measurements and packed-cell volume (PCV) were evaluated. Blood samples (n=12) were collected into one of four evacuated blood-collecting tubes: no anticoagulant (SER; yields serum), and plasma-yielding sodium heparin (HEP), sodium fluoride and potassium oxalate (NAF), and tripotassium ethylenediaminetetraacetic acid (K₃EDTA). Anticoagulant affected (P<0.05) metabolite values, with total cholesterol, triacylglycerol, and l-lactate highest in SER, and glucose highest in HEP; NEFA and β-hydroxybutyrate measurements were not affected (P>0.10) by anticoagulant. In addition, among the plasma-yielding tubes, PCV was highest in HEP and lowest in NAF (P<0.01). Experiment 2 investigated the effects of repetitive freezing-thawing cycles of plasma on metabolite levels. Blood samples (n=20) were collected using NAF tubes, and plasma was stored at −20 °C and thawed in a water bath (21 °C) 0, 1, 2, and 3 times within 18 h after collection. Compared with fresh samples (Thaw 0), by thaw 2, l-lactate increased (P<0.01) 5%, and glucose and total cholesterol decreased (P<0.001) 13 and 4%, respectively. Plasma NEFA increased 10% (P<0.01) between thaws 0 and 1, but returned to fresh levels (Thaw 0) with subsequent freeze-thaw cycles (P<0.05). Plasma β-hydroxybutyrate decreased (P<0.01) between thaws 0 and 1, but there was no further decline with subsequent freeze-thaw cycles (P<0.05). Experiment 3 evaluated the effects of plasma storage temperature (−20 °C versus −80 °C) and length (0–180 days) on metabolite levels in blood samples (n=12) collected in NAF tubes. All plasma metabolites were affected by storage length (day effect, P<0.01) but only total cholesterol values were affected by storage temperature, with values being higher in plasma stored at −20 than −80 °C (P<0.01). Glucose measurements were higher in samples stored at −20 °C for up to 30 days, but were higher thereafter in samples stored at −80 °C (storage length × temperature effect, P<0.01). Experiment 4 evaluated the effects of anticoagulant (SER versus NAF) and whole blood storage (4 °C) for 0, 1, 3, and 7 days on metabolite concentrations. Glucose was higher (P<0.0001) in NAF, possibly the result of the presence of the glycolytic inhibitor, sodium fluoride, whereas l-lactate, NEFA, total cholesterol and β-hydroxybutyrate were higher in SER (P<0.01). Total cholesterol, l-lactate, and NEFA increased, whereas β-hydroxybutyrate decreased with days in storage (P<0.01). Development of hemolysis in the samples artifactually elevated glucose and NEFA values by as much as 41 and 230%, respectively. Attention to proper blood handling, processing, and storage procedures, and avoidance of hemolysis are important in blood clinical analyses and in the proper interpretation of experimental results.     

Effects of seed maturation time and dry storage on light and temperature requirements during germination in invasive Prosopis juliflora

The effects of time of seed maturation and dry seed storage and of light and temperature requirements during seed incubation on final germination percentage and germination rate were assessed for the invasive shrub Prosopis juliflora (Sw.) D.C., grown under desert environmental conditions of the United Arab Emirates (UAE). Seeds were collected from Fujira on the northern coast of the UAE at different times during the growing seasons (autumn, winter and spring) and were germinated immediately and after 8 months of dry storage under room temperature (20±3 °C). Seeds were germinated at three temperatures (15, 25 and 40 °C) in both continuous light and darkness. The results showed significant effects for time of seed collection, seed storage, light and temperature of seed incubation and many of their interactions on both germination percentage and rate. Fresh seeds matured during autumn and winter germinated significantly greater at 40 °C and in light than at lower temperatures and in dark. Storage significantly increased germination percentage and rate; the increase was greater for seeds matured during winter than for seeds matured during spring. This indicates that dormancy breakage was greater in seeds of winter than seeds of spring. The need for high temperature to achieve greater germination was significantly reduced after seed storage, especially for seeds matured in autumn and winter.     

Ecophysiological requirements on seed germination of a Mediterranean perennial grass (Stipa tenacissima L.) under controlled temperatures and water stress

Stipa tenacissima L. (alpha grass) steppes are one of the most representative ecosystems in arid Mediterranean ecosystems. On the one hand these steppes, which are perpetually exposed to climate and strong anthropogenic pressure, have undergone severe degradation. On the other hand, the ability of S. tenacissima to regenerate naturally is significantly reduced. In this study the germination response and seedling emergence of S. tenacissima are examined in relation to the main environmental factors (water stress and temperature) under laboratory-controlled conditions. The main aim of this paper was to investigate the influence of temperature over a temperature range (10 °C–30 °C) and water stress induced by the solutions of polyethylene glycol (PEG)-6000 (0 to − 1.6 MPa) for a period of 30 days, on the germination behavior of S. tenacissima seeds. The results showed that temperatures between 10° and 20 °C seem to be favorable for the germination of this species, with optimum temperatures among accessions found in 20 °C. When seeds were water-stressed, germination severely decreased at − 0.8 MPa, indicating that the accession resistance limits to the water stress, and was completely inhibited at − 1.6 MPa. Consequently, the final germination percentage (FGP) decreased and the mean time germination (MTG) increased. Based on the empirical data of the germination rate, we estimated that the parameters of the thermal time and hydrotime models showed different values in all accessions which proves the difference between accession adaptive capacities.     

Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21 °C; control) and induced plasticity by rapid cold-hardening (RCH, 1 h at 0 °C followed by 1 h recovery), cold acclimation (CA, 5 days at 6 °C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between −16 and −23 °C, and were chill-susceptible. 80% of control flies were killed after 1 h at −7.2 °C (males) or −7.5 °C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0 °C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CT_min) at approximately −1.7 °C, which was ca. 0.5 °C colder than control flies; RCH and CA increased the CT_min compared to controls. Control and RCH flies exposed to 0 °C for 8 h took 30–40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.     

Microbial quality of common carp and pikeperch fingerlings cultured in a pond fed with treated wastewater

A pond with a surface area of 0.94 ha and a maximum depth of 1.6 m, situated on land owned by a wastewater treatment plant in Olsztynek (Poland), was filled with water from underground springs in the pond bottom. Twice during the production season, the pond was fed with sewage which had been biologically treated in sequencing batch reactors (SBR). No commercial feed or medicinal products were applied to the pond and natural feed in the pond was the only source of nutrition for the fish. After the production season, pikeperch and carp fingerlings (aged 0+), weighed 8.4 ± 1.2 g fish^?1 and 62.1 ± 13.6 g fish^?1, respectively.The microbiological examinations included tests of the pond water and the contents of the digestive tract, skin mucus and the muscles of the fingerlings. In the water the total cell count, heterotrophic bacteria count at a temperature of 22 °C and 37 °C (HPC 22 °C and 37 °C), coliforms, fecal coliforms, Streptococcus faecalis, Salmonella sp. and Aeromonas hydrophila, were recorded. The same groups of bacteria were noted in the analyzed fish tissue without the total cell count. In addition, the antibiotic sensitivity of A. hydrophila, isolated from all the examined environments, was tested.The low count of all examined groups of bacteria in the pond water was noted. The common carp tissues were more contaminated than the pikeperch tissues, but the microbial quality of both species was satisfactory. A. hydrophila strains proved to be highly sensitive to the tested antibiotics. The results of this study indicate that a high microbiological quality in the fingerlings was achieved thanks to the seasonal feeding of pond water by treated sewage.     

Effect of ambient temperature on female endurance performance

Ambient temperature can affect physical performance, and an ambient temperature range of −4 °C to 11 °C is optimal for endurance performance in male athletes. The few similar studies of female athletes appear to have found differences in response to cold between the genders. This study investigated whether ambient temperature affects female endurance performance. Nine athletes performed six tests while running on a treadmill in a climatic chamber at different ambient temperatures: 20, 10, 1, −4, −9 and −14 °C and a wind speed of 5 m s⁻¹. The exercise protocol consisted of a 10-min warm-up, followed by four 5-min intervals at increasing intensities at 76%, 81%, 85%, and 89% of maximal oxygen consumption. This was followed by an incremental test to exhaustion. Although peak heart rate, body mass loss, and blood lactate concentration after the incremental test to exhaustion increased as the ambient temperature rose, no changes in time to exhaustion, running economy, running speed at lactate threshold or maximal oxygen consumption were found between the different ambient temperature conditions. Endurance performance during one hour of incremental exercise was not affected by ambient temperature in female endurance athletes.     

Proteomic analysis of the adaptation to warming in the Antarctic bacteria Shewanella frigidimarina

Antarctica is subjected to extremely variable conditions, but the importance of the temperature increase in cold adapted bacteria is still unknown. To study the molecular adaptation to warming of Antarctic bacteria, cultures of Shewanella frigidimarina were incubated at temperatures ranging from 0 °C to 30 °C, emulating the most extreme conditions that this strain could tolerate. A proteomic approach was developed to identify the soluble proteins obtained from cells growing at 4 °C, 20 °C and 28 °C. The most drastic effect when bacteria were grown at 28 °C was the accumulation of heat shock proteins as well as other proteins related to stress, redox homeostasis or protein synthesis and degradation, and the decrease of enzymes and components of the cell envelope. Furthermore, two main responses in the adaptation to warm temperature were detected: the presence of diverse isoforms in some differentially expressed proteins, and the composition of chaperone interaction networks at the limits of growth temperature. The abundance changes of proteins suggest that warming induces a stress situation in S. frigidimarina forcing cells to reorganize their molecular networks as an adaptive response to these environmental conditions.     

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (< 50 °C), the enzymatic activity of the protein strongly decreased during storage at 60 °C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20 °C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.     

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2 °C, 5 °C or 8 °C water) and weekly, 1-h heat shocks (+3 °C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148 d at 2 °C, 92 d at 5 °C, 50 d at 8 °C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3 °C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8 °C and 5 °C embryos were significantly smaller and had larger yolks than 2 °C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.     

Impact of fluctuating temperatures on development of the koinobiont endoparasitoid Venturia canescens

The effect of temperature on the biology of Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae) is well understood under constant temperature conditions, but less so under more natural, fluctuating conditions. Herein we studied the influence of fluctuating temperatures on biological parameters of V. canescens. Parasitized fifth-instar larvae of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were reared individually in incubators at six fluctuating temperature regimes (15–19.5 °C with a mean of 17.6 °C, 17.5–22.5 °C with a mean of 19.8 °C, 20–30 °C with a mean of 22.7 °C, 22.5–27.5 °C with a mean of 25 °C, 25.5-32.5 °C with a mean of 28.3 °C and 28.5–33 °C with a mean of 30 °C) until emergence and death of V. canescens adults. Developmental time from parasitism to adult eclosion, adult longevity and survival were recorded at each fluctuating temperature regime. In principle, developmental time decreased with an increase of the mean temperature of the fluctuating temperature regime. Upper and lower threshold temperatures for total development were estimated at 34.9 and 6.7 °C, respectively. Optimum temperature for development and thermal constant were 28.6 °C and 526.3 degree days, respectively. Adult longevity was also affected by fluctuating temperature, as it was significantly reduced at the highest mean temperature (7.0 days at 30 °C) compared to the lowest one (29.4 days at 17.6 °C). Survival was low at all tested fluctuating temperatures, apart from mean fluctuating temperature of 25 °C (37%). Understanding the thermal biology of V. canescens under more natural conditions is of critical importance in applied contexts. Thus, predictions of biological responses to fluctuating temperatures may be used in population forecasting models which potentially influence decision-making in IPM programs.