The role of the heat shock proteins (HSP70 and sHSP) in the thermotolerance of freshwater amphipods from contrasting habitats

The aim of this study was to clarify the significance of HSP70 and sHSP for thermotolerance in freshwater amphipods. We compared four amphipod species from different freshwater habitats and biogeographical regions (Central Europe vs. Lake Baikal). Test individuals were exposed to thermal stress generated by a water temperature of 25 °C. The thermotolerance of the species, determined by median lethal time (LT50), followed in decreasing order by Gmelinoides fasciatus, Echinogammarus berilloni, Gammarus pulex, Eulimnogammarus verrucosus. HSP70 and sHSP base level concentrations for the species were determined at control (i.e. non-stress) conditions. For HSP70, the base levels were positively correlated to the species' thermotolerances. For sHSP, however, only thermotolerant G. fasciatus showed a high level. Thermal stress at 25 °C water temperature caused a deferred onset of HSP70 and sHSP expression followed by a subsequent offset, delineating a unimodal response curve. The time lag to the expression onset of HSP70 was shorter in the thermosensitive species, compared to thermotolerant ones. Conversely, the time span until the maximum level of HSP70 was variable, not showing a dependence on the thermotolerance properties of the species. The peak concentration in G. pulex was distinctly higher than in the other species, whereas E. verrucosus did not develop a well-defined response maximum at all. In sHSP, the temporal pattern of expression was even more variable than in HSP70. However, the thermosensitive species E. verrucosus showed a time lag of expression onset significantly shorter than the other species and thermotolerant G. fasciatus developed the most pronounced response maximum. Basing on these results, the cellular response to thermal stress in amphipods is more consistently reflected by HSP70, compared to sHSP.     

The effect of hyperthermia on cell viability,oxidative damage,and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The purpose of the study was to investigate the effects of mild hyperthermia on cell viability, release of lactate dehydrogenase (LDH), superoxide dismutase (SOD) activity, malondialdehyde (MDA) formation, total antioxidant capacity (T-AOC), and the relative mRNA levels of heat shock protein (HSP60, 70, and 90) in hepatic cells of grass carp (Ctenopharyngodon idellus) before and after temperature stress. Cultured cells were exposed to thermal stress (32 °C) for 0.5, 1, 2, 4, and 8 h. The results showed that hyperthermia stress significantly reduced cell viability (P<0.01) and increased LDH release at 0.5 and 1 h (P<0.05). Additionally, hyperthermia stress led to oxidative stress as evidenced by significantly decreased T-AOC after treating cells for 0.5 and 8 h (P<0.05). SOD activity also significantly decreased after 1 h of stress (P<0.05), but MDA formation increased after 8 h of stress (P<0.05). This may be partly responsible for the lower cell viability and higher LDH release we observed. The differences between SOD activity, MDA formation, and T-AOC between the 2 h treatment group and the control were smaller than that of other groups. This indicated that cellular antioxidant enzyme systems play an important role in the defense against oxidative stress. Further tests showed that the expression of HSP60 at 1, 2, and 4 h (P<0.05), HSP70 at 0.5 and 1 h (P<0.01), and HSP90 at all time points after stress were higher (P<0.01) than pre-stress levels. This suggested that HSPs possess the ability to modulate cellular anti-stress responses and play key roles in protecting organisms from heat stress. In conclusion, hyperthermia inhibits cell proliferation, induces cell oxidative stress, and enhances HSP expression in hepatic cells of grass carp.     

Testicular gene expression in male mice divergent for fertility after heat stress

Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35 ± 1 °C; n = 50) or thermoneutral (21 ± 1 °C; n = 10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n = 5), heat stress resistant (n = 5), and heat stress susceptible (n = 5) males, and 225 genes were observed to be differentially expressed (P < 0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using real-time RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31 ± 0.25); however, expression patterns of Canx and Tcp1 were not heritable.     

Heterologous expression of Anabaena PCC 7120 all3940 (a Dps family gene) protects Escherichia coli from nutrient limitation and abiotic stresses

This study presents first hand data on the cloning and heterologous expression of Anabaena PCC 7120 all3940 (a dps family gene) in combating nutrients limitation and multiple abiotic stresses. The Escherichia coli transformed with pGEX-5X-2-all3940 construct when subjected to iron, carbon, nitrogen, phosphorus limitation and carbofuron, copper, UV-B, heat, salt and cadmium stress registered significant increase in growth over the cells transformed with empty vector under iron (0%), carbon (0.05%), nitrogen (3.7 mM) and phosphorus (2 mM) limitation and carbofuron (0.025 mg ml⁻¹), CuCl₂ (1 mM), UV-B (10 min), heat (47 °C), NaCl (6% w/v) and CdCl₂ (4 mM) stress. Enhanced expression of all3940 gene measured by semi-quantitative RT-PCR at different time points under above mentioned treatments clearly demonstrates its role in tolerance against aforesaid abiotic stresses. This study opens the gate for developing transgenic cyanobacteria capable of growing successfully under above mentioned stresses.     

The timing of leaf fall affects cold acclimation by interactions with air temperature through water and carbohydrate contents

Three parameters (i.e. the water content, soluble sugar content and minimal air temperature) can be used to predict the cold acclimation process of walnut trees. In order to test this assumption, two-year-old walnuts were defoliated at two different dates, i.e. mechanical defoliation in early October (early leaf fall, EF) or natural defoliation in early November (natural leaf fall, NF) and conditioned in either outdoor freeze-deprived or cold-deprived (T_min > 13 °C) greenhouses over winter. Even if early defoliation date could have affected short day signal perception (SDSP), water balance and carbohydrate metabolism were more altered. EF treatment, by stopping transpiration, significantly increased tree's water content and at warm temperature high root activity stopped normal winter dehydration. Starch content decreased in all treatments, but there was only a significant increase in soluble sugar content when water content had sufficiently decreased. Thus, depending on date of defoliation, cold-deprived trees were or were not able to acclimate to frost (minimal frost hardiness = −21.8 °C vs. −22.1 °C in controls (freeze-deprived) for NF and −13.7 °C vs. −25.3 °C in controls for EF). Different treatments showed the relationship between minimal water content observed during winter and maximal soluble sugars synthesized. Thus, the cold acclimation process appeared dependent on these physiological parameters (water and soluble sugar contents) through the interaction between air temperature and timing of leaf fall.     

Effects of exposure to short-term heat stress on male reproductive fitness in a soil arthropod

Ambient temperature is a key environmental factor influencing a variety of aspects of the ecology and evolution of ectotherms. Reproductive traits have been suggested to be more sensitive to thermal stress than other life history traits. This study investigated the direct and indirect effects of heat shock on male reproductive success in the widespread springtail Orchesella cincta. Male springtails were exposed to four temperature treatments: heat hardening (35.2 °C for 1 h), heat shock (37.2 °C for 1 h), heat hardening + heat shock (35.2 °C for 1 h, followed 15 h later by 37.2 °C for 1 h), and control (20 °C). The heat shock gene Hsp70 showed high expression in all the heat treatments, indicating that the treatments indeed induced thermal stress. Significant mortality was only found in the treatment with heat shock, both with and without heat hardening. A direct effect of heat treatment was found on time to first reproduction, which was significantly longer after heat shock (with or without heat hardening) than in the control treatment. There was no difference among treatments in the number of spermatophores produced in the first reproductive instar. Heat treatment also had indirect effects on male reproductive success. Females chose significantly more spermatophores from control males than from males that received heat shock, heat hardening or both. A high percentage of spermatophores produced by heat shocked males caused reproductive failure in females, but no significant differences among treatments were found.Our results suggest that not all traits were equally affected by the heat stress. Heat hardening did not protect reproductive traits against the negative effects of heat shock. The indirect effects of heat shock on reproduction may be equally important as the direct effects.     

Analysis of midgut gene expression profiles from different silkworm varieties after exposure to high temperature

The silkworm is a poikilothermic animal, whose growth and development is significantly influenced by environmental temperature. To identify genes and metabolic pathways involved in the heat-stress response, digital gene expression analysis was performed on the midgut of the thermotolerant silkworm variety ‘932’ and thermosensitive variety ‘HY’ after exposure to high temperature (932T and HYT). Deep sequencing yielded 6,211,484, 5,898,028, 5,870,395 and 6,088,303 reads for the 932, 932T, HY and HYT samples, respectively. The annotated genes associated with these tags numbered 4357, 4378, 4296 and 4658 for the 932, 932T, HY and HYT samples, respecti'vely. In the HY-vs-932, 932-vs-932T, and HY-vs-HYT comparisons, 561, 316 and 281 differentially expressed genes were identified, which could be assigned to 179, 140 and 123 biological pathways, respectively. It was found that some of the biological pathways, which included oxidative phosphorylation, related to glucose and lipid metabolism, are greatly affected by high temperature and may lead to a decrease in the ingestion of fresh mulberry. When subjected to an early period of continuous heat stress, HSP genes, such as HSP19.9, HSP23.7, HSP40-3, HSP70, HSP90 and HSP70 binding protein, are up-regulated but then reduced after 24 h and the thermotolerant ‘932’ strain has higher levels of mRNA of some HSPs, except HSP70, than the thermosensitive variety during continuous high temperature treatment. It is suggested that HSPs and the levels of their expression may play important roles in the resistance to high temperature stress among silkworm varieties. This study has generated important reference tools that can be used to further analyze the mechanisms that underlie thermotolerance differences among silkworm varieties.     

Transcriptional profiling of mouse liver in response to chronic heat stress

Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 °C) or control (24 °C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

Multigenerational analysis of temperature and salinity variability affects on metabolic rate,generation time,and acute thermal and salinity tolerance in Daphnia pulex

Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. Increasing levels of environmental stresses are known to induce the cellular stress response (CSR). Energy for the CSR may be provided by an elevated overall metabolic rate. However, if metabolic rate is constant or lower under elevated stress, energy for the CSR is taken from other physiological processes, such as growth or reproduction. This study investigated the examined energetic responses to the combination of temperature and salinity variability during a multigenerational exposure of partheogenetically reproducing Daphnia pulex. We raised D. pulex in an orthogonal combination of daily fluctuations in temperature (15, 15–25, 15–30 °C) and salinity (0, 0–2, 0–5). Initially metabolic rates were lower under all variable temperature and variable salinity treatments. By the 6th generation there was little metabolic variation among low and intermediate temperature and salinity treatments, but metabolic suppression persisted at the most extreme salinity. When grown in the control condition for the 6th generation, metabolic suppression was only observed in D. pulex from the most extreme condition (15–30 °C, 0–5 salinity). Generation time was influenced by acclimation temperature but not salinity and was quickest in specimens reared at 15–25 °C, likely due to Q₁₀ effects at temperatures closer to the optima for D. pulex, and slowest in specimens reared at 15–30 °C, which may have reflected elevated CSR. Acute tolerance to temperature (LT₅₀) and salinity (LC₅₀) were both highest in D. pulex acclimated to 15–30 °C and salinity 0. LT₅₀ and LC₅₀ increased with increasing salinity in specimens raised at 15 °C and 15–25 °C, but decreased with increasing salinity in specimens raised at 15–30 °C. Thus, increasing temperature confers cross-tolerance to salinity stress, but the directionality of synergistic effects of temperature and salinity depend on the degree of environmental variability. Overall, the results of our study suggest that temperature is a stronger determinant of metabolism, growth, and tolerance thresholds, and assessment of the ecological impacts of environmental change requires explicit information regarding the degree of environmental variability.     

Heat shock protein 70 response to physical and chemical stress in Chamelea gallina

Intertidal area is characterized by several fluctuations in natural agents and anthropogenic factors (oxygen levels, temperature, salinity, B[a]P presence) that cause a noticeable increase in the expression rate of heat shock protein 70 (HSP70). HSPs acting as molecular chaperones and their induction represent a specific cellular defence mechanism in response to several stress.Chamelea gallina specimens from the North Adriatic coast were exposed to different experimental conditions: varying oxygen levels (48 h of anoxia followed by 24 h of normoxic recovery), temperatures (20, 25, 30 °C for 7 days), salinity (28, 34, 40‰ for 7 days) and B[a]P concentrations (0.5 mg/L for 24 h, 7 and 12 days). Following the extraction of the digestive gland and gills, HSP70 levels were identified in the cytosolic fraction by immunoblotting using primary monoclonal antibodies. An increase in the rate of HSP70 expression under anoxic conditions in the digestive gland was observed at high temperatures, at low salinity and in the presence of B[a]P. The protein was overexpressed in the absence of oxygen and after 12 days of B[a]P exposure, while it was underexpressed in hyposaline conditions in the gills.HSP70 induction can be considered an adaptation mechanism associated with changes in environmental parameters, but also with xenobiotic presence. The overexpression of HSP70 is therefore induced by protein damage due to stressogenic factors. HSP recruitment renders them available for the processes of folding and refolding of denatured proteins or for their transport to a degradation system. The evident sensitivity of HSP70 to natural and anthropogenic stressogenic agents was examined in the present research.The results of this research revealed an interesting response of heat shock protein 70 in C. gallina, underlining the sensitivity of this important commercial species to natural and anthropogenic stress agents.