Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Genomic,evolutionary and expression profile analysis of <Emphasis Type="Italic">Hsp70</Emphasis> superfamily in A and D genome of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp.) under the challenge of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

In this study, we comparatively analyzed the 115 Hsp70 genes identified in Gossypium raimondii, Gossypium hirsutum and Gossypium arboreum genomes. Those Hsp70 genes unequally distributed among chromosomes in A and D genome of cotton (Gossypium spp.), and were classified into 29 groups according to the homology of them. Based on the localization information of the orthologs in Arabidopsis, the Hsp70 proteins were predicted to locate in cytosol, endoplasmic reticulum, mitochondrion or chloroplast. Homologous analysis indicated the evolutionary conservation of Hsp70 in cotton. In addition, those Hsp70 genes were differently expressed in Suyuan-045, Hai-7124 and TM-1, which were highly resistant, resistant, and sensitive to Verticillium dahliae respectively. The expressions of 26 Hsp70 genes were induced by Verticillium dahliae except for Hsp70-07/16/25/26, and the result suggested the potential involvement of them in responding to Verticillium wilt. Hsp70-08/30/31 was highly expressed in both Suyuan-045 and Hai-7124, and it was hypothesized that they might be involved in the resistance to the invasion of Verticillium dahliae. 144h after inoculation with Verticillium dahliae, the expression of Hsp70-13/14/15 was only up-regulated in Suyuan-045, and it was assumed that they might be involved in resistance to the extension of Verticillium dahliae. Further study on those Hsp70 genes would be valuable to reveal the role of them in Verticillium wilt resistance.     

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.     

Heat shock proteins in <Emphasis Type="Italic">Varroa destructor</Emphasis> exposed to heat stress and in-hive acaricides

Varroa destructor is one of the major pests that affect honeybees around the world. Chemical treatments are common to control varroosis, but mites possess biochemical adaptive mechanisms to resist these treatments, enabling them to survive. So far, no information is available regarding whether these pesticides can induce the expression of heat shock protein (Hsp) as a common protective mechanism against tissue damage. The aims of this study were to determine differences in heat shock tolerance between mites collected from brood combs and phoretic ones, and to examine patterns of protein expression of Hsp70 that occur in various populations of V. destructor after exposure to acaricides commonly employed in beekeeping, such as flumethrin, tau-fluvalinate and coumaphos. Curiously, mites obtained from brood cells were alive at 40 °C, unlike phoretic mites that reached 100% mortality, demonstrating differential thermo-tolerance. Heat treatment induced Hsp70 in mites 4?×?more than in control mites and no differences in response were observed in phoretic versus cell-brood-obtained mites. Dose–response assays were carried out at increasing acaricide concentrations. Each population showed a different stress response to acaricides despite belonging to the same geographic region. In one of them, coumaphos acted as a hormetic stressor. Pyrethroids also induced Hsp70, but mite population seemed sensitive to this treatment. We concluded that Hsp70 could represent a robust biomarker for measuring exposure of V. destructor to thermal and chemical stress, depending on the acaricide class and interpopulation variability. This is relevant because it is the first time that stress response is analyzed in this biological model, providing new insight in host-parasite-xenobiotic interaction.     

The Effect of Acute Hiperthermia on Levels of Hsp70 and Lipid Peroxidation in Laboratory-Cultured Pulmonate Mollusk <Emphasis Type="Italic">Stagnicola corvus</Emphasis>

This study evaluates dynamic changes in levels of Hsp70s and products of lipid peroxidation due to hyperthermia in the laboratory-raised freshwater pulmonate mollusk Stagnicola corvus (Gmelin, 1791). It is concluded that acute hyperthermia in S. corvus leads to the activation of nonspecific mechanisms of stress adaptation, which is manifested in an elevated Hsp70s level in the beginning of exposure, followed by a reduction in protein concentration, and the further preservation of the Hsp70 at a level exceeding the constitutive forms. At the same time, the level of LPO products tends to decrease, which indicates strengthening of the antioxidant defense processes in response to stress.     

Ayurvedic <Emphasis Type="Italic">Amalaki Rasayana</Emphasis> promotes improved stress tolerance and thus has anti-aging effects in <Emphasis Type="BoldItalic">Drosophila melanogaster</Emphasis>

Amalaki Rasayana (AR) is a common Ayurvedic herbal formulation of Phyllanthus emblica fruits and some other ingredients, and is used for general good health and healthy aging. We reported it to improve life history traits and to suppress neurodegeneration as well as induced apoptosis in Drosophila. The present study examines responses of Drosophila reared on AR-supplemented food to crowding, thermal or oxidative stresses. Wild-type larvae/flies reared on AR-supplemented food survived the various cell stresses much better than those reared on control food. AR-fed mutant park ¹³ or DJ-1β ^Delta93 (Parkinson’s disease model) larvae/flies, however, showed only partial or no protection, respectively, against paraquat-induced oxidative stress, indicating essentiality of DJ-1β for AR-mediated oxidative stress tolerance. AR feeding reduced the accumulation of reactive oxygen species (ROS) and lipid peroxidation even in aged (35-day-old) wild-type flies while enhancing superoxide dismutase (SOD) activity. We show that while Hsp70 or Hsp83 expression under normal or stress conditions was not affected by AR feeding, Hsp27 levels were elevated in AR-fed wild-type control as well as heat-shocked larvae. Therefore, besides the known anti-oxidant activity of Phyllanthus emblica fruits, dietary AR also enhances cellular levels of Hsp27. Our in vivo study on a model organism shows that AR feeding significantly improves tolerance to a variety of cell stresses through reduced ROS and lipid peroxidation on the one hand, and enhanced SOD activity and Hsp27 on the other. The resulting better homeostasis improves life span and quality of organism’s life.     

Identification of a drought responsive gene encoding a nuclear protein involved in drought and freezing stress tolerance in Arabidopsis

Plants have developed adaptive strategies to survive under different abiotic stressors. To identify new components involved in abiotic stress tolerance, we screened unannotated expressed sequence tags (ESTs) and evaluated their cold or drought response in Arabidopsis. We identified a drought response gene (DRG) encoding a 39.5-kDa polypeptide. This protein was expressed specifically in siliques and was induced by drought stress in most tissues. When a DRG-GFP construct was introduced into Arabidopsis protoplasts, GFP signals were detected only in the nucleus. The drg mutant plant was more sensitive to mannitol-induced osmotic stress in agar plates and to drought or freezing stress in soil than the wild-type. Activating the DRG restored the normal sensitivity of drg mutants to abiotic stressors. No differences in drought or freezing tolerance were observed between the wild-type and transgenic plants overexpressing the DRG. When DRG was expressed in a cold-sensitive Escherichia coli strain BX04, the transformed bacteria grew faster than the untransformed BXO4 cells under cold stress. These results demonstrate that DRG is a nuclear protein induced by abiotic stresses and it is required for drought and freezing tolerance in Arabidopsis.     

Association between circulating levels of heat-shock protein 27 and aggressive periodontitis

Heat-shock protein (Hsp) 27 is a major intracellular molecular chaperone and controller of intracellular responses to inflammatory signals. In the extracellular space, recombinant Hsp27 has been described to exert anti-inflammatory activities. The aim of this study was to assess the association between circulating levels of Hsp27 and different types of periodontitis. Pro- and anti-inflammatory cytokines and the stress proteins Hsp27 and Hsp60 with proposed anti- and pro-inflammatory properties, respectively, were measured by two-site ELISA in the serum of patients with aggressive periodontitis (AgP, n?=?30), chronic periodontitis (CP, n?=?29) and periodontally healthy controls (H, n?=?28). Furthermore, Hsp27 and Hsp60 levels were also measured longitudinally in 12 AgP patients at 6 time points up to 3 months after treatment. AgP patients had lower levels of Hsp27 compared to CP patients and healthy subjects (adjusted one-way ANOVA, p?<?0.001, followed by post hoc Tukey HSD comparisons), while no differences in levels of Hsp60 or cytokines between the three groups were detected. In CP patients and H subjects, the systemic Hsp27 levels correlated with Hsp60 (r?=?0.43, p?<?0.001; r?=?0.59, p?<?0.001, respectively) and with pro-inflammatory cytokines TNF-α (r?=?0.48, p?<?0.001; r?=?0.55, p?<?0.001, respectively) and IL-6 (r?=?0.44, p?<?0.01). However, no such correlations were detected in AgP cases. No consistent temporal patterns of changes of Hsp27 concentration were detected across AgP patients following periodontal treatment. This study provides the first evidence that Hsp27 may be differentially expressed and regulated in AgP patients as compared with CP patients and healthy individuals.     

Characterization and Expression Dynamics of Key Genes Involved in the Gilthead Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) Cortisol Stress Response during Early Ontogeny

The present study identified and characterized six key genes involved in the hypothalamic-pituitary-interrenal (HPI) axis of gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. The key genes involved in the HPI axis for which gene structure and synteny analysis was carried out, comprised of two functional forms of glucocorticoid receptors (GR), as well as three forms of pro-opiomelanocortin (POMC) genes and one form of mineralocorticoid receptor (MR) gene. To explore their functional roles during development but also in the stress response, the expression profiles of gr1, gr2, mr, pomc_aI, pomc_aII, and pomc_β were examined during early ontogeny and after an acute stress challenge. The acute stress challenge was applied at the stage of full formation of all fins, where whole body cortisol was also measured. Both the cortisol and the molecular data implied that sea bream larvae at the stage of the full formation of all fins at 45 dph are capable of a response to stress of a similar profile as observed in adult fish.