Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: Effect of temperature variability

It is considered that extreme environmental temperature, rather than mean temperatures exert a selective pressure in ectotherms. Consequently, it is important to understand how the predicted increase in temperature variance with a higher frequency of extreme events in climate change is likely to impact on organisms. Thermal tolerance traits (i.e. chill-coma, recovery time, Hsp70 expression) are directly linked with performance in ectotherms and have consequences in life-history traits. We examined the effects of temperature variability on thermal tolerance and life-history traits through ontogeny of an insect with a complex life-cycle: the yellow mealworm beetle Tenebrio molitor. We established two common gardens with 100 recently ovoposited eggs each. Larvae were reared from hatching to adult on either a variable (mean=18 °C and a variance of 6.8 °C) or constant (18±1 °C) thermal environment. Development rate and growth rate were similar between thermal environments. Results indicate that larvae reared in a variable environment are more cold-tolerant than larvae of a constant environment. Interestingly, these results are reversed in the adult stage, outlining an inter-stage physiological cost. Gene expression pattern of an Hsp70 gene was well correlated with larval thermotolerance to cold in the variable environment but higher gene expression in adults is not correlated with individual's thermotolerance. We conclude that chill-coma, recovery time and Hsp70 gene expression are plastic in response to a thermal environment but also change significantly their responses depending on the ontogenetic stage, implying that the response of adult individuals is linked to early stages of the life-cycle.     

Effects of heat stress on ovarian development and the expression of HSP genes in mice

Heat stress reduces oocyte competence, thereby causing lower fertility in animals. Chronic and acute heat stresses cause extensive morphological damage in animals, but few reports have focused on the effects of chronic and acute heat stresses on ovarian function and heat shock protein (HSP) gene expression during ovarian injury. In this study, we subjected female mice to chronic and acute heat stresses; we then calculated the ovary index, examined ovary microstructure, and measured the expression of multiple HSP family genes. Chronic heat stress reduced whole-body and ovarian growth but had little effect on the ovarian index; acute heat stress did not alter whole-body or ovarian weight. Both chronic and acute heat stresses impaired ovary function by causing the dysfunction of granular cells. Small HSP genes increased rapidly after heat treatment, and members of the HSP40, HSP70, and HSP90 families were co-expressed to function in the regulation of the heat stress response. We suggest that the HSP chaperone machinery may regulate the response to heat stress in the mouse ovary.     

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.     

Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens

Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs) are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h ²) and the additive genetic coefficient of variation (CV _A) of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes) and extracellular (serum) protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R), the h ² and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h ² and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CV _A of 13.3–16.2% in young adults and of 2.7–8.1% in pre-harvest adults. Thus, despite its low h ², HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals.     

Isolation of four heat shock protein cDNAs from grapefruit peel tissue and characterization of their expression in response to heat and chilling temperature stresses

In order to continuously supply horticultural products for long periods, it is essential to store them after harvest in low temperatures. However, many tropical and subtropical fruits and vegetables, such as citrus, are sensitive to chilling. In previous studies, the authors have shown that a short hot water rinsing treatment (at 62°C for 20 s) increased chilling tolerance in grapefruit. In order to gain more insight into the molecular mechanisms involved in heat‐induced chilling tolerance, PCR cDNA subtraction analysis was performed which isolated four different PCR fragments whose expression was enhanced 24 h after the heat treatment, and that showed high sequence homology with various plant HSP18‐I, HSP18‐II, HSP22 and HSP70 genes. It was found that the short hot water treatment given at 62°C for 20 s, but not at lower temperatures of 20 or 53°C, increased the expression of the various HSP cDNAs in grapefruit peel tissue. However, when the fruits were kept at ambient temperatures, the increases in HSP mRNA levels following the hot water treatment were temporary and lasted only between 6 and 48 h. Similar temporary increases in the HSP mRNA levels were detected following exposure of the fruit to a hot air treatment at 40°C for 2 h. Nevertheless, when the fruits were treated with hot water but afterwards stored at chilling temperatures of 2°C, the mRNA levels of the various HSP18‐I, HSP18‐II, HSP22 and HSP70 cDNAs increased and remained high and stable during the entire 8‐week cold‐storage period, suggesting their possible involvement in heat‐induced chilling‐tolerance responses. The chilling treatment by itself increased the expression of the HSP18‐I cDNA, but had no effect on the mRNA levels of any of the other HSP cDNAs. Exposure of fruit to other stresses, such as wounding, UV irradiation, anaerobic conditions and exposure to ethylene, had no effect on the expression of the various HSPs. Overall, the study explored the correlation between the expression and persistence of various HSP cDNAs in grapefruit peel tissue during cold storage, on the one hand, and the acquisition of chilling tolerance, on the other hand, and the results suggest that HSPs may play a general role in protecting plant cells under both high‐ and low‐temperature stresses.     

The Stress of Suicide: Temporal and Spatial Expression of Putative Heat Shock Protein 70 Protect the Cells from Heat Injury in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Heat stress adversely affects growth, development, and yield of winter wheat (Triticum aestivum). Plants have, however, evolved mechanisms to adapt to such conditions mainly by the expression of stress-associated chaperones, the heat shock proteins (HSPs), for modulating the tolerance level. Here, we report cloning of cytosolic putative HSP70 of 1678 bp from a thermotolerant cultivar (C306) of wheat (T. aestivum). A BLASTn search showed maximum homology with the predicted HSP70 protein reported from Hordeum vulgare (accession no AK354795.1). In silico characterization showed the presence of a nucleotide-binding domain of the sugar kinase/HSP70/actin superfamily in the sequence. Putative HSP70 showed temporal and spatial variations in the expression under heat stress (HS). We observed the abundance of HSP70 protein, H₂O₂, proline, and guaiacol peroxidase activity during the seed-hardening stage under HS; accumulation was, however, higher in the thermotolerant C306 than in thermosusceptible HD2329 cultivar. A gradual decrease in cell membrane stability (CMS) and an increase in total antioxidant capacity (TAC) were observed in both the cultivars at the different stages of growth. The expression of HSP70 showed a negative correlation with CMS and a positive correlation with TAC under HS; changes were less pronounced in C306 than in HD2329 at all the stages of growth studied. HSP70 seems to play diverse roles associated with thermotolerance, and partially protect wheat from terminal HS. Being the important member of family of the HSPs, HSP70 needs to be studied in detail, to be used for developing climate-smart wheat crops, through genetic engineering/breeding approaches.     

A novel small molecule HSP90 inhibitor,NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1^G93A similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca²⁺ accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1^G93A transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.     

Molecular characterization and gene evolution of the heat shock protein 70 gene in snakehead fish with different tolerances to temperature

Temperature tolerances (including lethal limits) and associated rates of thermal acclimation of fish are critical information in predicting fish responses to global climate changes. In this study, a partial sequence of the heat shock protein 70 gene (HSP70) from the fish species Channa striatus was isolated and characterized. Evolutionary process that led to the diversity of HSP70 specific to vertebrates was also analysed. Results revealed that HSP70 is highly homologous in other fish families. The conservation of the HSP 70 gene among fish families could be driven by forces of natural selection due to climatic change. We exposed C. striatus to heat shock (32 °C) and cold shock (16 °C) respectively, in order to examine the differences of temperatures in influencing the expression patterns of HSP70. We revealed that expression of HSP70 was higher at 32 °C than at 16 °C in most of the organs. Specifically, occurrence of chaperone activity of HSP70 was found at low temperature. Therefore, this fish was postulated that to seems to be able to survive at lower temperature compared to higher temperature indicating there is force of natural selection acting towards this HSP 70 gene. This will demonstrate the effect of global warming towards the fish survivability.     

外源亚精胺对淹涝胁迫下黄瓜根HSP70表达的影响

采用逆转录聚合酶链式反应(RT-PCR)及蛋白免疫印迹杂交(Western Blot)技术,研究0.5 mmol/L亚精胺浸种的黄瓜幼苗在淹水胁迫下,根热激蛋白70基因(HSP70)mRNA和蛋白质的表达量的变化。结果表明:淹水胁迫使黄瓜根HSP70的mRNA和蛋白的表达呈现先上升后下降的趋势,在淹水4 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理; 亚精胺浸种的黄瓜根HSP70的mRNA和蛋白的表达量在24 h内呈一直上升的趋势,在淹水24 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理。淹涝胁迫下,亚精胺浸种的黄瓜根HSP70的mRNA和蛋白表达量在淹水12 h和24 h时极显著高于蒸馏水浸种。外源亚精胺能诱导淹涝胁迫下黄瓜幼苗根HSP70 mRNA和蛋白质的表达量的增加,缓解淹涝胁迫对黄瓜造成的伤害。     

Characterization of HSP70 expression in drones of Apis cerana (Hymenoptera: Apidae)

Heat shock proteins (HSPs) constitute a superfamily of molecular chaperones that are rapidly biosynthesized in response to various biotic and abiotic factors. In this study, we first cloned the full-length HSP70 gene of the Eastern honeybee Apis cerana. Then, using real-time quantitative PCR, we explored HSP70 expression profiles in drones at different developmental stages, ages, and reproductive statuses (with and without semen). The full-length HSP70 cDNA is 2421 bp, including a 1953-bp open reading frame (ORF) that encodes a polypeptide of 650 amino acids. The HSP70 gene consists of one intron and two exons. The phylogenetic analysis revealed that the HSP70 genes of A. cerana and Apis mellifera are the most closely related. We observed HSP70 expression at all selected developmental stages and detected the highest expression in pupae with an unpigmented body cuticle and brown eyes (Pb) and much lower expression in larvae hatched within 72 h. In adult drones of different ages, the highest expression level of HSP70 was observed in 16-day-old drones; significantly lower accumulation of HSP70 mRNA was detected in 4-day-old drones. There was no significant difference in HSP70 expression between drones with and without semen captured at the entrance, while the HSP70 gene expression level strikingly differed between drones captured at the entrance and the drones collected within the hive. Our study suggests that HSP70 might play a critical role in drone development and during reproductive mating events.     

Contributions of Dopamine-Related Genes and Environmental Factors to Highly Sensitive Personality: A Multi-Step Neuronal System-Level Approach

Traditional behavioral genetic studies (e.g., twin, adoption studies) have shown that human personality has moderate to high heritability, but recent molecular behavioral genetic studies have failed to identify quantitative trait loci (QTL) with consistent effects. The current study adopted a multi-step approach (ANOVA followed by multiple regression and permutation) to assess the cumulative effects of multiple QTLs. Using a system-level (dopamine system) genetic approach, we investigated a personality trait deeply rooted in the nervous system (the Highly Sensitive Personality, HSP). 480 healthy Chinese college students were given the HSP scale and genotyped for 98 representative polymorphisms in all major dopamine neurotransmitter genes. In addition, two environment factors (stressful life events and parental warmth) that have been implicated for their contributions to personality development were included to investigate their relative contributions as compared to genetic factors. In Step 1, using ANOVA, we identified 10 polymorphisms that made statistically significant contributions to HSP. In Step 2, these polymorphism''s main effects and interactions were assessed using multiple regression. This model accounted for 15% of the variance of HSP (p<0.001). Recent stressful life events accounted for an additional 2% of the variance. Finally, permutation analyses ascertained the probability of obtaining these findings by chance to be very low, p ranging from 0.001 to 0.006. Dividing these loci by the subsystems of dopamine synthesis, degradation/transport, receptor and modulation, we found that the modulation and receptor subsystems made the most significant contribution to HSP. The results of this study demonstrate the utility of a multi-step neuronal system-level approach in assessing genetic contributions to individual differences in human behavior. It can potentially bridge the gap between the high heritability estimates based on traditional behavioral genetics and the lack of reproducible genetic effects observed currently from molecular genetic studies.     

Heat shock protein 9 mRNA expression increases during fruiting body differentiation in Grifola frondosa and other edible mushrooms

We isolated a homolog of Schizosaccharomyces pombe HSP9 from Grifola frondosa and designated it Gf.HSP9. The Gf.HSP9 gene consisted of four exons and three introns and encoded 84 amino acid residues. We have also identified related HSP9 genes from Pleurotus eryngii (Pe.HSP9), Hypsizygus marmoreus (Hm.HSP9), and Lentinula edodes (Le.HSP9). The predicted tertiary structures of these HSP9s were the same. In addition, the expression pattern of their mRNA increased during fruiting body in a differentiation-dependent manner. These results suggested that HSP9 was associated with the fruiting body differentiation in these four edible mushrooms.     

Estimating the Potential for Adaptation of Corals to Climate Warming

The persistence of tropical coral reefs is threatened by rapidly increasing climate warming, causing a functional breakdown of the obligate symbiosis between corals and their algal photosymbionts (Symbiodinium) through a process known as coral bleaching. Yet the potential of the coral-algal symbiosis to genetically adapt in an evolutionary sense to warming oceans is unknown. Using a quantitative genetics approach, we estimated the proportion of the variance in thermal tolerance traits that has a genetic basis (i.e. heritability) as a proxy for their adaptive potential in the widespread Indo-Pacific reef-building coral Acropora millepora. We chose two physiologically different populations that associate respectively with one thermo-tolerant (Symbiodinium clade D) and one less tolerant symbiont type (Symbiodinium C2). In both symbiont types, pulse amplitude modulated (PAM) fluorometry and high performance liquid chromatography (HPLC) analysis revealed significant heritabilities for traits related to both photosynthesis and photoprotective pigment profile. However, quantitative real-time polymerase chain reaction (qRT-PCR) assays showed a lack of heritability in both coral host populations for their own expression of fundamental stress genes. Coral colony growth, contributed to by both symbiotic partners, displayed heritability. High heritabilities for functional key traits of algal symbionts, along with their short clonal generation time and high population sizes allow for their rapid thermal adaptation. However, the low overall heritability of coral host traits, along with the corals'' long generation time, raise concern about the timely adaptation of the coral-algal symbiosis in the face of continued rapid climate warming.     

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558_HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558_HS expressing cytoplasmic HSP70, and purified EXO_HSP and EXO_HS from J558_HSP and J558_HS tumour cell culture supernatants by ultracentrifugation. We found that EXO_HSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Ia^b, CD40, CD80 and inflammatory cytokines than EXO_HS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXO_HSP are able to stimulate type 1 CD4⁺ helper T (Th1) cell responses, and more efficient P1A‐specific CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO_HS. In addition, we further elucidate that EXO_HSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8⁺ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.     

Systematic analysis of HSP gene expression and effects on cell growth and survival at high hydrostatic pressure in Saccharomyces cerevisiae

We systematically investigated the role of HSP genes in the growth and survival of Saccharomyces cerevisiae under high hydrostatic pressure together with analysis of pressure-regulated gene expression. Cells of strain BY4742 were capable of growth at moderate pressure of 25 MPa. When pressure of 25 MPa was applied to the cells, the expression of HSP78, HSP104, and HSP10 was upregulated by about 3- to 4-fold, and that of HSP32, HSP42, and HSP82 was upregulated by about 2- to 2.6-fold. However, the loss of one of the six genes did not markedly affect growth at 25 MPa, while the loss of HSP31 impaired high-pressure growth. These results suggest that Hsp31 plays a role in high-pressure growth but that the six upregulated genes do not. Extremely high pressure of 125 MPa decreased the viability of the wild-type cells to 1% of the control level. Notably, the loss of HSP genes other than HSP31 enhanced the survival rate by about fivefold at 125 MPa, suggesting that the cellular defensive system against high pressure could be strengthened upon the loss of the HSP genes. In this paper, we describe the requirement for and significance of a subset of HSP genes in yeast cell growth at moderate pressure and survival at extremely high pressure.     

ENVIRONMENT-DEPENDENCE OF QUANTITATIVE GENETIC PARAMETERS IN IMPATIENS PALLIDA

Population response to selection depends on the presence of additive genetic variance for traits under selection. When a population enters an alien environment, environment-induced changes in the expression of genetic variance may occur. These could have large effects on the response to selection. To investigate the environment-dependence of genetic variance, we conducted a reciprocal transplant experiment between two ecotypically differentiated populations of Impatiens pallida using the progeny of a standard mating design. The floodplain site was characterized by high water availability, moderate temperatures, and continuous dense stands of Impatiens. The hillside site was drier, with larger temperature extremes and supported only scattered patches of Impatiens with significantly lower seed production and earlier mortality. Estimates of heritability were low for each of the 13 traits measured in each population and site (range from 0–28%). Additive genetic variance for life-history traits tended to be larger than for morphological traits, but genetic variance in fitness was estimated to be not significantly different from zero in all cases. Significant heritability was detected in both populations for one trait (date of first cleistogamous flower) known to be closely related to fitness on the hillside. In general, heritability was reduced for populations when grown in the hillside site relative to the floodplain site, suggesting that stress acts to reduce the expression of genetic variance and the potential to respond to selection there. Consistent reductions in heritability associated with more stressful environments suggest that populations invading such sites may undergo little adaptive differentiation and be more prone to local extinction.