Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

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.     

Two small heat shock protein genes in Apis cerana cerana: characterization,regulation, and developmental expression

In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4 °C) and heat shock (37 °C and 43 °C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms.     

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.     

Distinct chemical contaminants induce the synthesis of Hsp70 proteins in green microalgae Desmodesmus subspicatus: Heat pretreatment increases cadmium resistance

Using western-blotting techniques, we examined the effect of differently acting contaminants, such as anthracene (PAH), cadmium (heavy metal) and chloridazone (herbicide), as well as heat shock on the production of two Hsp70 proteins (cytoplasmic and stromal) in planktonic algae Desmodesmus subspicatus. All contaminants applied stimulated production of both Hsp70s in a concentration-dependent manner, but heat shock treatment turned out to be the most effective. Heat shock pretreatment (for 1 h at 40 °C) induced tolerance to cadmium in algal cells (measured by changes in growth rate), but not to anthracene or chloridazone. Two Hsp70s from D. subspicatus cells representing cytoplasmic and stromal proteins were purified by ATP-affinity chromatography.     

Immunological enhancement action of endotoxin-free tilapia heat shock protein 70 against Streptococcus iniae

The immunological effects of heat shock proteins (HSPs) had been found in humans and mice, but scarce data of endotoxin-free Hsp70 were reported in tilapia. In the current study, we reported that tHsp70 alone and antigen-tHsp70 compound increased the proliferations of lymphocytes and macrophages, significantly increased the NO release and phagocytotic ability of macrophages (p < 0.05), and enhanced the levels of immune-related genes in lymphocytes and macrophages in a dose- and/or time-dependent manner. On the other hand, tHsp70 not only helped to reduce the proliferation inhibitions induced by the ECP treatment, but also assisted antigens to enhance the vaccine-induced protection against Streptococcus iniae (p < 0.05). We described, for the first time, a critical role of endotoxin-free tHsp70 on activation of tilapia lymphocytes and macrophages post S. iniae exposure and its up-regulation effects on vaccine-induced protection. Our research highlights the immunological enhancement action of Hsp70 in teleost immunity.     

siRNA-mediated knockdown of CiGRP78 gene expression leads cell susceptibility to heavy metal cytotoxicity

Heavy metal ion is one of the critical environmental pollutants accumulated in living organisms and causes toxic or carcinogenic effects once passed threshold levels. As an important member of Hsp70 (heat shock protein 70) family, the 78-kDa glucose-regulated protein (GRP78) can enhance cell survival rates remarkably under thermal stress. Recent studies also demonstrated that the expression of GRP78 enhances the cell survival under heavy metal stress. In this study, three most representative heavy metal ions, Pb^2 +, Hg^2 + and Cd^2 +, were used to stimulate Ctenopharyngodon idella kidney (CIK) cells. The results showed that cell viability under Pb^2 +, Hg^2 + and Cd^2 + stress decreased significantly. The longer and the greater the concentrations of stimulation from heavy metal ions, the higher the rate of cell death was observed. Among them, Hg^2 + is the most hazardous to cells. Under the same stress condition, Hg^2 + resulted in 50% of cell death, Cd^2 + (or Pb^2 +) led to 45% (or 35%) of cell death, respectively. Western immunoblotting indicated that C. idella GRP78 (CiGRP78) protein expression level was enhanced obviously in CIK cells under Pb^2 +, Hg^2 + and Cd^2 + stress, meaning CiGRP78 is involved in heavy metal cytotoxicity. To further study the role of CiGRP78 in cytoprotection, we designed the siRNA against CiGRP78 (from nucleotides + 788 to + 806) and transfected it into CIK cells to silence endogenous CiGRP78. The viability rate of CIK cells transfected with or without siRNA incubated with HgCl₂ for 12 h showed a significant decrease from 50% to 21%. Our results showed that CiGRP78 protects cells against heavy metal stimuli to some extent.     

Thermal tolerance,net CO2 exchange and growth of a tropical tree species, Ficus insipida, cultivated at elevated daytime and nighttime temperatures

Global warming and associated increases in the frequency and amplitude of extreme weather events, such as heat waves, may adversely affect tropical rainforest plants via significantly increased tissue temperatures. In this study, the response to two temperature regimes was assessed in seedlings of the neotropical pioneer tree species, Ficus insipida. Plants were cultivated in growth chambers at strongly elevated daytime temperature (39 °C), combined with either close to natural (22 °C) or elevated (32 °C) nighttime temperatures. Under both growth regimes, the critical temperature for irreversible leaf damage, determined by changes in chlorophyll a fluorescence, was approximately 51 °C. This is comparable to values found in F. insipida growing under natural ambient conditions and indicates a limited potential for heat tolerance acclimation of this tropical forest tree species. Yet, under high nighttime temperature, growth was strongly enhanced, accompanied by increased rates of net photosynthetic CO₂ uptake and diminished temperature dependence of leaf-level dark respiration, consistent with thermal acclimation of these key physiological parameters.