Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Effect of the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment

It has been demonstrated that precooling with ice slurry ingestion enhances endurance exercise capacity in the heat. However, no studies have yet evaluated the optimal timing of ice slurry ingestion for precooling. This study aimed to investigate the effects of varying the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment. Ten active male participants completed 3 experimental cycling trials to exhaustion at 55% peak power output (PPO) after 15 min of warm-up at 30% PPO at 30 °C and 80% relative humidity. Three experimental conditions were set: no ice slurry ingestion (CON), pre-warm-up ice slurry ingestion (−1 °C; 7.5 g kg⁻¹) (PRE), and post-warm-up ice slurry ingestion (POST). Rectal and mean skin temperatures at the beginning of exercise in the POST condition (37.1±0.2 °C, 33.8±0.9 °C, respectively) were lower than those in the CON (37.5±0.3 °C; P<0.001, 34.8±0.8 °C; P<0.01, respectively) and PRE (37.4±0.2 °C; P<0.01, 34.6±0.7 °C; P<0.01, respectively) conditions. These reductions increased heat storage capacity and resulted in improved exercise capacity in the POST condition (60.2±8.7 min) compared to that in the CON (52.0±11.9 min; effect size [ES]=0.78) and PRE (56.9±10.4 min; ES=0.34) conditions. Ice slurry ingestion after warm-up effectively reduced both rectal and skin temperatures and increased cycling time to exhaustion in a warm environment. Timing ice slurry ingestion to occur after warm-up may be effective for precooling in a warm environment.     

Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed ‘thermal performance curve’. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.     

Pre-cooling with intermittent ice ingestion lowers the core temperature in a hot environment as compared with the ingestion of a single bolus

The timing in which ice is ingested may be important for optimizing its success. However, the effects of differences in the timing of ice ingestion has not been studied in resting participants. Therefore, the purpose of this study was to investigate the effects of differences in the timing of ice ingestion on rectal temperature (Tre) and rating of perceptual sensation in a hot environment. Seven males ingested 1.25 g kg⁻¹ of crushed ice (ICE1.25: 0.5 °C) or cold water (CON: 4 °C) every 5 min for 30 min, or were given 7.5 g kgBM⁻¹ of crushed ice (ICE7.5) to consume for 30 min in a hot environment (35 °C, 30% relative humidity). The participants then remained at rest for 1 h. As physiological indices, Tre, body mass and urine specific gravity were measured. Rating of thermal sensation was measured at 5-min intervals throughout the experiment. ICE1.25 continued to decrease Tre until approximately 50 min, and resulted in a greater reduction in Tre (−0.56±0.20 °C) than ICE7.5 (−0.41±0.14 °C). Tre was reduced from 40 to 75 min by ICE1.25, which is a significant reduction in comparison to ICE7.5 (p<.05). Mean RTS with ICE1.25 at 50–65 min was significantly lower than that with ICE7.5 (p<.05). These results suggest that pre-cooling with intermittent ice ingestion is a more effective strategy both for lowering the Tre and for the rating of thermal sensation.     

Thermal tolerance and preference of exploited turbinid snails near their range limit in a global warming hotspot

Predicted global climate change has prompted numerous studies of thermal tolerances of marine species. The upper thermal tolerance is unknown for most marine species, but will determine their vulnerability to ocean warming. Gastropods in the family Turbinidae are widely harvested for human consumption. To investigate the responses of turbinid snails to future conditions we determined critical thermal maxima (CTMax) and preferred temperatures of Turbo militaris and Lunella undulata from the tropical-temperate overlap region of northern New South Wales, on the Australian east coast. CTMax were determined at two warming rates: 1 °C/30 min and 1 °C/12 h. The number of snails that lost attachment to the tank wall was recorded at each temperature increment. At the faster rate, T. militaris had a significantly higher CTMax (34.0 °C) than L. undulata (32.2 °C). At the slower rate the mean of both species was lower and there was no significant difference between them (29.4 °C for T. militaris and 29.6 °C for L. undulata). This is consistent with differences in thermal inertia possibly allowing animals to tolerate short periods at higher temperatures than is possible during longer exposure times, but other mechanisms are not discounted. The thermoregulatory behaviour of the turban snails was determined in a horizontal thermal gradient. Both species actively sought out particular temperatures along the gradient, suggesting that behavioural responses may be important in ameliorating short-term temperature changes. The preferred temperatures of both species were higher at night (24.0 °C and 26.0 °C) than during the day (22.0 °C and 23.9 °C). As the snails approached their preferred temperature, net hourly displacement decreased. Preferred temperatures were within the average seasonal seawater temperature range in this region. However, with future predicted water temperature trends, the species could experience increased periods of thermal stress, possibly exceeding CTMax and potentially leading to range contractions.     

Temperature-dependent development of Lista haraldusalis (Walker) (Lepidoptera: Pyralidae) on Platycarya strobilacea

The effect of constant temperatures on development and survival of Lista haraldusalis (Walker) (Lepidoptera: Pyralidae), a newly reported insect species used to produce insect tea in Guizhou province (China), was studied in laboratory conditions at seven temperatures (19 °C, 22 °C, 25 °C, 28 °C, 31 °C, 34 °C, and 37 °C) on Platycarya strobilacea. Increasing the temperature from 19 °C to 31 °C led to a significant decrease in the developmental time from egg to adult emergence, and then the total developmental time increased at 34 °C. Egg incubation was the stage where L. haraldusalis experienced the highest mortality at all temperatures. The survival of L. haraldusalis was significantly higher at 25 °C and 28 °C, whereas none of the eggs hatched at 37 °C. Common and Ikemoto linear models were used to describe the relationship between the temperature and the developmental rate for each immature stage of L. haraldusalis. The estimated values of the lower temperature threshold and thermal constant of the total immature stages using Common and Ikemoto linear models were 11.34 °C and 11.20 °C, and 939.85 and 950.41 degree-days, respectively. Seven nonlinear models were used to fit the experimental data to estimate the developmental rate of L. haraldusalis. Based on the biological significance for model evaluation, Ikemoto linear, Logan-6, and SSI were the best models that fitted each immature stage of L. haraldusalis and they were used to estimate the temperature thresholds. These thermal requirements and temperature thresholds are crucial for facilitating the development of factory-based mass rearing of L. haraldusalis.     

Comparison between ambulatory measurement of effective thermal conductivity and laser Doppler flowmetry method to assess skin microcirculatory activity

The main objective of this paper was to assess the performance of the ambulatory device μHematron to measure indirectly skin blood flow relative to the well-established Laser Doppler flowmetry method. The μHematron device is dedicated to the non-invasive measurement of effective thermal conductivity of living tissues, based on the thermal clearance method. Its major advantage is its ambulatory functionality, as available methods for evaluation of microcirculatory activity are non-ambulatory methods. An experiment was conducted on ten healthy women exposed for one hour in three different thermal environments (22 °C, 25 °C and 30 °C). Skin microcirculatory activity was analyzed after an acclimatization period of 30 minutes. The time between each exposure was at least one hour. Performances of the μHematron device were assessed and a comparative study with a laser Doppler perfusion monitor (LDPM) was performed. Good correlation coefficients between the two devices (r = 0.71 at T1 = 22 °C, r = 0.77 at T2 = 25 °C and r = 0.83 at T3 = 30 °C) were obtained while the LDPM signal was filtered by a low pass filter (0.1 Hz). These results showed that continuous monitoring of effective thermal conductivity was possible in neutral and warm ambiences. Then, the μHematron device could be considered as a complementary tool to Doppler techniques for the investigation of skin blood flow, when ambulatory conditions are required.     

Development response of Spodoptera exigua to eight constant temperatures: Linear and nonlinear modeling

Temperature-dependent development of Spodoptera exigua (Hübner) were evaluated at eight constant temperatures of 12, 15, 20, 25, 30, 33, 34 and 36 °C with a variation of 0.5 °C on sugar beet leaves. No development occurred at 12 °C and 36 °C. Total developmental time varied from 120.50 days at 15 °C to 14.50 days at 33 °C. As temperature increased from 15 °C to 33 °C, developmental rate (1/developmental time) of S. exigua increased but declined at 34 °C. The lower temperature threshold (T_min) was estimated to be 12.98 °C and 12.45 °C, and the thermal constant (K) was 294.99 DD and 311.76 DD, using the traditional and Ikemoto–Takai linear models, respectively. The slopes of the Ikemoto–Takai linear model for different immature stages were different, violating the assumption of rate isomorphy. Data were fitted to three nonlinear models to predict the developmental rate and estimate the critical temperatures. The T_min values estimated by Lactin-2 (12.90 °C) and SSI (13.35 °C) were higher than the value estimated by Briere-2 (8.67 °C). The estimated fastest development temperatures (T_fast) by the Briere-2, Lactin-2 and SSI models for overall immature stages development of S. exigua were 33.4 °C, 33.9 °C and 32.4 °C, respectively. The intrinsic optimum temperature (T_Φ) estimated from the SSI model was 28.5 °C, in which the probability of enzyme being in its native state is maximal. The upper temperature threshold (T_max) values estimated by these three nonlinear models varied from 34.00 °C to 34.69 °C. These findings on thermal requirements can be used to predict the occurrence, number of generations and population dynamics of S. exigua.     

Clostridium difficile survives minimal temperature recommended for cooking ground meats

We quantified the thermal inhibitory effect of 71 °C (recommended for cooking ground meats), and re-heating at 85 °C, on food- and food-animal-derived Clostridium difficile spores. All C. difficile strains tested (n = 20) survived 71 °C for 2 h, but 90% died within 10 min when re-heated at 85 °C. Current cooking recommendations would need revision to include C. difficile.     

Effect of rearing temperature on growth and thermal tolerance of Schizothorax (Racoma) kozlovi larvae and juveniles

Effect of rearing temperature on growth and thermal tolerance of Schizothorax (Racoma) kozlovi Nikolsky larvae and juveniles was investigated. The fish (start at 12 d post hatch) were reared for nearly 6 months at five constant temperatures of 10, 14, 18, 22 and 26 °C. Then juvenile fish being acclimated at three temperatures of 14, 18 and 22 °C were chosen to determine their critical thermal maximum (CTMax) and lethal thermal maximum (LTMax) by using the dynamic method. Growth rate of S. kozlovi larvae and juveniles was significantly influenced by temperature and fish size, exhibiting an increase with increased rearing temperature, but a decline with increased fish size. A significant ontogenetic variation in the optimal temperatures for maximum growth were estimated to be 24.7 °C and 20.6 °C for larvae and juveniles of S. kozlovi, respectively. The results also demonstrated that acclimation temperature had marked effects on their CTMax and LTMax, which ranged from 32.86 °C to 34.54 °C and from 33.79 °C to 34.80 °C, respectively. It is suggested that rearing temperature must never rise above 32 °C for its successful aquaculture. Significant temperature effects on the growth rate and thermal tolerance both exhibit a plasticity pattern. Determination of critical heat tolerance and optima temperature for maximum growth of S. kozlovi is of ecological significance in the conservation and aquaculture of this species.     

Biology and thermal requirements of Chrysoperla genanigra (Neuroptera: Chrysopidae) reared on Sitotroga cerealella (Lepidoptera: Gelechiidae) eggs

Chrysoperla genanigra Freitas is a common green lacewing associated with melon pests in the Northeastern Brazil. All life stages of this recently described species were studied under a range of constant temperature conditions (17, 21, 25, 29, 33, 35 and 37 °C), a photoperiod of 12 h:12 h (L:D) and 70 ± 10% relative humidity. Adults of C. genanigra were fed on a diet consisting of a 1:1 (v/v) mixture of brewer’s yeast and honey, while larvae were provided with eggs of Sitotroga cerealella (Olivier) ad libitum. The duration of preimaginal development of the species was inversely proportional to temperature and ranged from approximately 63 days at 17 °C to 15 days at 35 °C. The percentage of adult emergence varied from 6.7% at 17 °C to 76.7% at 25 °C, although no larvae were able to complete development at 37 °C. The lower thermal threshold for total preimaginal development was approximately 10.8 °C and the thermal requirement was 336.7 degree-days. Egg production, along with the longevity of both males and females, were significantly affected by temperature. It is concluded that the best temperature for rearing C. genanigra is 25 °C, with the lowest preimaginal mortality and the highest egg production (992.7 eggs/female).     

Isolation and Characterization of Antifreeze Proteins from the Antarctic Marine Microalga Pyramimonas gelidicola

Antifreeze proteins (AFPs) play an important role in the psychrophilic adaptation of polar organisms. AFPs encoded by an Antarctic chlorophyte, identified as Pyramimonas gelidicola, were isolated and characterized. Two AFP isoforms were found from cDNAs and their deduced molecular weights were estimated to be 26.4 kDa (Pg-1-AFP) and 27.1 kDa (Pg-2-AFP). Both AFP cDNAs were cloned and expressed in Escherichia coli. The purified recombinant Pg-1-rAFP and Pg-2-rAFP both showed antifreeze activity based on the measurement of thermal hysteresis (TH) and morphological changes to single ice crystals. Pg-1-rAFP shaped ice crystals into a snowflake pattern with a TH value of 0.6?±?0.02 °C at ~15 mg/ml. Single ice crystals in Pg-2-rAFP showed a dendritic morphology with a TH value of 0.25?±?0.02 °C at the same protein concentration. Based on in silico protein structure predictions, the three-dimensional structures of P. gelidicola AFPs match those of their homologs found in fungi and bacteria. They fold as a right-handed β-helix flanked by an α-helix. Unlike the hyperactive insect AFPs, the proposed ice-binding site on one of the flat β-helical surfaces is neither regular nor well-conserved. This might be a characteristic of AFPs used for freeze tolerance as opposed to freeze avoidance. A role for P. gelidicola AFPs in freeze tolerance is also consistent with their relatively low TH values.     

Comparison of the thermal performance between a population of the olive fruit fly and its co-adapted parasitoids

Long-term separation of a host from its native parasitoids may result in divergent thermal adaptation between host and parasitoid. The olive fruit fly, Bactrocera oleae (Rossi), most likely originated from Sub-Saharan Africa, but has since had a long invasion history in cultivated olives that spans geographical barriers and continents. This study compared three major thermal performance profiles (development, survival, and reproduction) across a wide range of temperatures (10–34 °C) among a Californian population of the olive fruit fly and two African parasitoids, Psyttalia lounsburyi (Silvestri) and Psyttalia humilis (Silvestri), believed to have co-adapted with the fruit fly in its native range. Temperature ranges for the development and survival were 10–30 °C for the fly, 10–28 °C for P. lounsburyi, and 14–32 °C for P. humilis. There was no difference in any thermal performance measured between two P. humilis populations (Kenya and Namibia) tested. The most suitable temperature ranges for reproduction were 22–30 °C for the fly, 18–32 °C for P. humilis, and 18–26 °C for P. lounsburyi. The results showed slight differences in the thermal profiles among olive fruit fly and both parasitoids species, with P. humilis being more heat tolerant whereas P. lounsburyi was less heat tolerant than the fruit fly. The results are discussed with respect to thermal co-adaptation and classical biological control of the olive fruit fly.     

Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.)

To test the hypothesis that impaired mitochondrial respiration limits cardiac performance at warm temperatures, and examine if any effect(s) are sex-related, the consequences of high temperature on cardiac mitochondrial oxidative function were examined in 10 °C acclimated, sexually immature, male and female Atlantic cod. Active (State 3) and uncoupled (States 2 and 4) respiration were measured in isolated ventricular mitochondria at 10, 16, 20, and 24 °C using saturating concentrations of malate and pyruvate, but at a submaximal (physiological) level of ADP (200 µM). In addition, citrate synthase (CS) activity was measured at these temperatures, and mitochondrial respiration and the efficiency of oxidative phosphorylation (P:O ratio) were determined at [ADP] ranging from 25–200 µM at 10 and 20 °C. Cardiac morphometrics and mitochondrial respiration at 10 °C, and the thermal sensitivity of CS activity (Q₁₀=1.51), were all similar between the sexes. State 3 respiration at 200 µM ADP increased gradually in mitochondria from females between 10 and 24 °C (Q₁₀=1.48), but plateaued in males above 16 °C, and this resulted in lower values in males vs. females at 20 and 24 °C. At 10 °C, State 4 was ~10% of State 3 values in both sexes [i.e. a respiratory control ratio (RCR) of ~10] and P:O ratios were approximately 1.5. Between 20 and 24 °C, State 4 increased more than State 3 (by ~70 vs. 14%, respectively), and this decreased RCR to ~7.5. The P:O ratio was not affected by temperature at 200 μM ADP. However, (1) the sensitivity of State 3 respiration to increasing [ADP] (from 25 to 200 μM) was reduced at 20 vs. 10 °C in both sexes (K_m values 105±7 vs. 68±10 μM, respectively); and (2) mitochondria from females had lower P:O values at 25 vs. 100 μM ADP at 20 °C, whereas males showed a similar effect at 10 °C but a much more pronounced effect at 20 °C (P:O 1.05 at 25 μM ADP vs. 1.78 at 100 μM ADP). In summary, our results demonstrate several sex-related differences in ventricular mitochondrial function in Atlantic cod, and suggest that myocardial oxidative function and possibly phosphorylation efficiency may be limited at temperatures of 20 °C or above, particularly in males. These observations could partially explain why cardiac function in Atlantic cod plateaus just below this species׳ critical thermal maximum (~22 °C) and may contribute to yet unidentified sex differences in thermal tolerance and swimming performance.     

Behavioral thermoregulation and critical thermal limits of giant keyhole limpet Megathura crenulata (Sowerby 1825) (Mollusca; Vetigastropoda)

The thermoregulatory behavior of the giant keyhole limpet Megathura crenulata was determined in a horizontal thermal gradient during the day at 18.9 °C and 18.3 °C for the night. The final preferendum determined for giant keyhole limpets was of 18.6±1.2 °C.Limpets' displacement velocity was 10.0±3.9 cm h⁻¹ during the light phase and 8.4±1.6 cm h⁻¹ during the dark phase. The thermotolerance (measured as CTMax at 50%) was determined in a keyhole limpet in three acclimation temperatures 17, 20, and 23 °C. Limpets were subjected to water increasing temperatures at a rate of 1 °C every 30 min, until they detached from the substrate. The critical thermal maximum at 50% was 27.2, 27.9 and 28.3 °C respectively.     

Thermal tolerance,growth and oxygen consumption of Labeo rohita fry (Hamilton, 1822) acclimated to four temperatures

A 30 day feeding trial was conducted using a freshwater fish, Labeo rohita (rohu), to determine their thermal tolerance, oxygen consumption and optimum temperature for growth. Four hundred and sixteen L. rohita fry (10 days old, 0.385±0.003 g) were equally distributed between four treatments (26, 31, 33 and 36 °C) each with four replicates for 30 days. Highest body weight gain and lowest feed conversion ratio (FCR) was recorded between 31 and 33 °C. The highest specific growth rate was recorded at 31 °C followed by 33 and 26 °C and the lowest was at 36 °C. Thermal tolerance and oxygen consumption studies were carried out after completion of growth study to determine tolerance level and metabolic activity at four different acclimation temperatures. Oxygen consumption rate increased significantly with increasing acclimation temperature. Preferred temperature decided from relationship between acclimation temperature and Q₁₀ values were between 33 and 36 °C, which gives a better understanding of optimum temperature for growth of L. rohita. Critical thermal maxima (CTMax) and critical thermal minima (CTMin) were 42.33±0.07, 44.81±0.07, 45.35±0.06, 45.60±0.03 and 12.00±0.08, 12.46±0.04, 13.80±0.10, 14.43±0.06, respectively, and increased significantly with increasing acclimation temperatures (26, 31, 33 and 36 °C). Survival (%) was similar in all groups indicating that temperature range of 26–36 °C is not fatal to L. rohita fry. The optimum temperature range for growth was 31–33 °C and for Q₁₀ values was 33–36 °C.     

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.     

Behavioral and mortality responses of the bivalves Scrobicularia plana and Cerastoderma edule to temperature,as indicator of climate change's potential impacts

Temperature is one of the most important abiotic factors affected by climate change. It determines physiological processes, ecological patterns and establishes the limits of geographic distribution of species. The induced thermal stress frequently results in physiological and behavioral responses and, in extreme cases, may lead to mortality episodes. Scrobicularia plana and Cerastoderma edule behavioral and mortality responses to temperature were evaluated. Specimens were sampled in the Mondego estuary (Portugal), acclimated and exposed to different temperature treatments (5–35 °C). Individual activity and mortality were registered during 120 h laboratory assays. Both species showed a thermal optimum for their activity (S. plana: 15–23 °C; C. edule: 20–23 °C), and survival was mainly affected by high temperature (S. plana: LC50_120 h = 28.86 °C; C. edule: LC50_120 h = 28.01 °C), with 100% mortality above critical values (≥32 °C). Results further indicated that both species are more affected the higher the temperature and the longer the exposure time. This study indicates that the occurrence of extreme climatic events, especially heat waves, may be particularly impairing for these species.     

The effect of acclimation temperature on thermal activity thresholds in polar terrestrial invertebrates

In the Maritime Antarctic and High Arctic, soil microhabitat temperatures throughout the year typically range between ?10 and +5 °C. However, on occasion, they can exceed 20 °C, and these instances are likely to increase and intensify as a result of climate warming. Remaining active under both cool and warm conditions is therefore important for polar terrestrial invertebrates if they are to forage, reproduce and maximise their fitness. In the current study, lower and upper thermal activity thresholds were investigated in the polar Collembola, Megaphorura arctica and Cryptopygus antarcticus, and the mite, Alaskozetes antarcticus. Specifically, the effect of acclimation on these traits was explored. Sub-zero activity was exhibited in all three species, at temperatures as low as ?4.6 °C in A. antarcticus. At high temperatures, all three species had capacity for activity above 30 °C and were most active at 25 °C. This indicates a comparable spread of temperatures across which activity can occur to that seen in temperate and tropical species, but with the activity window shifted towards lower temperatures. In all three species following one month acclimation at ?2 °C, chill coma (=the temperature at which movement and activity cease) and the critical thermal minimum (=low temperature at which coordination is no longer shown) occurred at lower temperatures than for individuals maintained at +4 °C (except for the CTmin of M. arctica). Individuals acclimated at +9 °C conversely showed little change in their chill coma or CTmin. A similar trend was demonstrated for the heat coma and critical thermal maximum (CTmax) of all species. Following one month at ?2 °C, the heat coma and CTmax were reduced as compared with +4 °C reared individuals, whereas the heat coma and CTmax of individuals acclimated at +9 °C showed little adjustment. The data obtained suggest these invertebrates are able to take maximum advantage of the short growing season and have some capacity, in spite of limited plasticity at high temperatures, to cope with climate change.