Cold resistance in two species of cave-dwelling beetles (Coleoptera: Cholevidae)

Supercooling points (SCPs), lower lethal temperatures (LLTs), and the effect of short-term exposures (1 min) to low temperatures were examined in the adults of two stenothermal leptodirin species, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae). Specimens were collected from two caves in the Venetian Prealps (NE-Italy). Inter-species comparison highlighted lower values of SCP in N. mancinii (−7.1±0.9 °C) than in N. pasai (−6.4±0.3 °C), with no significant intersexual differences in both species. N. pasai (LLT₅₀±SE=−16.96±2.30 °C; LLT₁₀₀=−25.41 °C) tolerated short exposures to subzero temperatures better than N. mancinii (LLT₅₀±SE=−4.89±1.08 °C; LLT₁₀₀=−11.72 °C). According to the mortality and cumulative proportion of individual freezing curves (CPIF), SCPs and LLT₁₀₀, N. pasai may be defined as “strongly freeze tolerant”, N. mancinii as “moderately freezing tolerant”. Overall, these results may justify the different in-cave habitat selection showed by the two species (N. pasai was abundant close to the entrance where the temperature is variable whereas N. mancinii was confined to the internal part of the cave where the temperature is constant throughout the year), and suggest hypotheses on the effects of such habitat selection on freeze tolerance strategy adopted. Finally, they give new insights into possible responses to climate changes in cave dwelling species.     

Protein expression during heat stress in thermo-intolerant and thermo-tolerant diatoms

Diatoms (Chrysophyta) are photosynthetic microorganisms that are abundant in the natural environment and often associated with specific habitat and water quality conditions. Their significance as bioindicators and as exploitable sources of fine chemicals makes them desirable candidates for the study of stress responses. The protein expression of a thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) diatom following exposure to elevated temperature was investigated using one- and two-dimensional gel electrophoresis and Western blot analysis. It was determined using SDS PAGE with ³⁵S-methionine labeled proteins and Western blot analysis using pea HSP70 antisera that higher temperatures and longer duration treatment were required to cause a noticeable stress response in C. muelleri compared to P. tricornutum. This may be explained by C. muelleri possessing higher amounts of constitutively expressed heat shock proteins, which allows these cells to rapidly adjust to temperature increases. Two-dimensional gel electrophoresis revealed that putative small heat shock proteins (smHSPs) may appear to play a role during heat stress in both diatoms, which is similar to the response in plants. SDS PAGE data are also presented characterizing the recovery of P. tricornutum after heat shock. These results suggest that there is a lag period between heat shock and stress protein synthesis in these thermo-intolerant cells. This supports the hypothesis that cells without higher amounts of constitutively expressed stress proteins have a greater sensitivity to increased temperature. Work is underway to identify particular stress proteins responsible for conveying thermo-tolerance and to determine if overexpression of these genes in thermo-intolerant diatoms affects their temperature sensitivity.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

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.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.