Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Regulation of heat shock proteins in the apple maggot Rhagoletis pomonella during hot summer days and overwintering diapause

Abstract Developing larvae of the apple maggot Rhagoletis pomonella are frequently exposed to summertime apple temperatures that exceed 40 °C and, during their overwintering diapause, pupae are exposed to sub‐zero soil temperatures for prolonged periods. To investigate the potential involvement of heat shock proteins (Hsps) in response to these environmental extremes, the genes encoding Hsp70 and Hsp90 in R. pomonella are cloned and expression monitored during larval feeding within the apple and during overwintering pupal diapause. Larvae reared in the laboratory at constant temperatures of 25, 28 or 35 °C express Hsp90 but very little Hsp70. Larvae do not survive rearing at 40 °C. The temperature cycles to which larvae were exposed inside apples in the field, ranging 16–46.9 °C over a 24‐h period, elicit strong Hsp70 and Hsp90 expression, which begins at mid‐day and reaches a peak in late afternoon, coinciding with peak air and apple temperatures. Heat shock proteins are also expressed strongly by pupae during their overwintering diapause. Hsp70 is not expressed in nondiapausing pupae but is highly expressed throughout diapause. Hsp90 is constitutively expressed in both diapausing and nondiapausing pupae. Rhagoletis pomonella thus strongly expresses its Hsps during pupal diapause, presumably as a protection against low temperature injury, and during larval development to cope with natural temperature cycles prevailing in late summer.     

induction of heat shock response and acquisition of thermotolerance in callus cultures of Gerbera jamesonii

Summary The heat shock (HS) response in callus cultures of the ornamental plant Gerbera jamesonii H. Bolus var. hybrida was analyzed. A HS at 35° C or 40° C for 4 h induced (a) the synthesis of several heat shock proteins (HSPs), especially in the small molecular weight range and some spots corresponding to HSP70 components, and (b) an increase in the steady state levels of some specific mRNAs. At the nonstressing temperature (26° C), a sustainable level of translation for HSP70 was indeed carried out, as confirmed by immunological analysis with a monoclonal antibody against cotton HSP70. The steady state levels of mRNAs measured before and after a HS by Northern hybridization showed an increase with the heterologous probes HSP17.4, HSP17.6, and HSP21, whereas the probes HSC70 and HSP70 did not show any difference between the levels of control and HS-mRNAs. A pretreatment at 35° C, which induced a set of HSPs in the callus cultures, decreased the cell damage upon exposure to a temperature of 45° C as determined either with a regrowth test or by the tetrazolium reduction assay. Typically, as with the whole plants, callus of Gerbera jamesonii possessed the ability to respond to HS both by inducing HSPs and by developing an acquired thermotolerance.     

The effects of population and thermal acclimation on the growth,condition and cold responsive mRNA expression of age-0 lake sturgeon (Acipenser fulvescens)

In Manitoba, Canada, wild lake sturgeon (Acipenser fulvescens) populations exist along a latitudinal gradient and are reared in hatcheries to bolster threatened populations. We reared two populations of lake sturgeon, one from each of the northern and southern ends of Manitoba and examined the effects of typical hatchery temperatures (16°C) as well as 60-day acclimation to elevated rearing temperatures (20°C) on mortality, growth and condition throughout early development. Additionally, we examined the cold shock response, which may be induced during stocking, through the hepatic mRNA expression of genes involved in the response to cold stress and homeoviscous adaptation (HSP70, HSP90a, HSP90b, CIRP and SCD). Sturgeon were sampled after 1 day and 1 week following stocking into temperatures of 8, 6 and 4°C in a controlled laboratory environment. The southern population showed lower condition and higher mortality during early life than the northern population while increased rearing temperature impacted the growth and condition of developing northern sturgeon. During the cold shock, HSP70 and HSP90a mRNA expression increased in all sturgeon treatments as stocking temperature decreased, with higher expression observed in the southern population. Expression of HSP90b, CIRP and SCD increased as stocking temperature decreased in northern sturgeon with early acclimation to 20°C. Correlation analyses indicated the strongest molecular relationships were in the expression of HSP90b, CIRP and SCD, across all treatments, with a correlation between HSP90b and body condition in northern sturgeon with early acclimation to 20°C. Together, these observations highlight the importance of population and rearing environment throughout early development and on later cellular responses induced by cold stocking temperatures.     

Arabidopsis HSP70‐16 is required for flower opening under normal or mild heat stress temperatures

Sepals play important roles in protecting inner floral organs from various stresses and in guaranteeing timely flower opening. However, the exact role of sepals in coordinating interior and exterior signals remains elusive. In this study, we functionally characterized a heat shock protein gene, Arabidopsis HSP70‐16, in flower opening and mild heat stress response, using combined genetics with anatomic, physiological, chemical, and molecular analyses. We showed that HSP70‐16 is required for flower opening and mild heat response. Mutation of HSP70‐16 led to a significant reduction in seed setting rate under 22°C, which was more severe at 27°C. Mutation of HSP70‐16 also caused postgenital fusion at overlapping tips of two lateral sepals, leading to failed flower opening, abnormal floral organ formation, and impaired fertilization and seed setting. Chemical and anatomic analyses confirmed specific chemical and morphological changes of cuticle property in mutant lateral sepals, and qRT‐PCR data indicated that expression levels of different sets of cuticle regulatory and biosynthetic genes were altered in mutants grown at both 22°C and 27°C temperatures. This study provides a link between thermal and developmental perception signals and expands the understanding of the roles of sepal in plant development and heat response.     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

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.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

Genome‐wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress

Heat shock proteins (HSP)are essential molecular chaperones that play important roles in the stress stimulation of insects.Bemisia tabaci,a phloem feeder and invasive species,can cause extensive crop damage through direct feeding and transmission of plant viruses.Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome.In total,we identified 26 Hsp genes,including three Hsp90,17 Hsp70,one Hsp60 and five sHSP (small heat shock protein)genes.The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here.The gene structures among the same families are relatively conserved.Meanwhile,the motif compositions and secondary structures of BtHsp proteins were predicted.In addition,quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies.Most Hsp genes were induced or repressed by thermal stress (40℃)and cold treatment (4℃)in whitefly.Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45℃.All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions.Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.     

Effects of rearing and induction temperature on the temporal dynamics of heat shock protein 70 expression in a butterfly

The temporal dynamics of heat shock protein 70 (HSP70) expression in response to longer‐term acclimation and rapid hardening in the butterfly Lycaena tityrus is investigated. After a 1‐h exposure to 1 °C or 37 °C, HSP70 is quickly up‐regulated within 1 h and down‐regulated within 2 h. The fast dynamic of HSP70 expression is in contrast to the patterns found in organisms inhabiting more stable thermal environments, and is interpreted as an adaptation to the large and rapid temperature variation experienced by flying ectotherms. HSP70 expression is higher in males than in females, as well as in animals reared at 27 °C than at 20 °C, although it is very similar across the high and low induction temperatures. Animals reared at the higher temperature, however, respond less strongly to high‐temperature stress.     

Calcium/calmodulin-dependent protein kinase II of the oriental fruit fly,Bactrocera dorsalis,and its association with rapid cold hardiness

The oriental fruit fly, Bactrocera dorsalis, is a serious insect pest with diverse host range. Furthermore, its invasive and polyphagous behaviors allow this species to expand its habitats. Recent climate change and increase of international trade/transportation facilitate the species expansion from subtropical to temperate regions. Low temperature during winter appears to be the major factor limiting its expansion to temperate zones in the northern hemisphere. This study reports its remarkable ability in rapid cold-hardening (RCH) along with deep supercooling capacity. A brief exposure to 9?°C significantly enhanced cold tolerance of its larvae, pupae, and adults. RCH took 1–2?h for pupae and adults, although it took 24?h for larvae. Supercooling capacity of pupae was also enhanced by RCH treatment from ?13.4?°C to ?16.6?°C. To trace genetic factors associated with RCH, calcium/calmodulin-dependent protein kinase II (Bd-CaMKII) was identified from B. dorsalis and their expression in response to RCH treatment was analyzed. Bd-CaMKII possesses three conserved domains of kinase, calmodulin, and oligomerization. Bd-CaMKII is highly homologous to CaMKII of D. melanogaster and other tephritid flies. Expression levels of Bd-CaMKII in the larvae treated with RCH were significantly increased by approximately 5.5 folds compared to those in control larvae. In addition, expression levels of HSP70 and HSP90 were also increased in response to RCH treatment. These results along with previous studies suggest that cold-hardening of B. dorsalis is functionally associated with its supercooling capacity with increased production of cryoprotectants and HSP through regulatory activity of Bd-CaMKII.     

Temporal variation in constitutive and inducible heat shock proteins in the Zebra Finch Taeniopygia guttata

Cellular stressors initiate the heat shock response mediated by heat shock proteins (HSPs). There are two main types of HSPs, constitutive (always expressed) and inducible (upon stress), but as many in vivo studies fail to distinguish between them and because temporal expression patterns often differ among various types of HSPs, it is unclear when to measure HSPs. In this study, 26 (13 per treatment) adult female Zebra Finches Taeniopygia guttata were heat‐stressed (39 °C) or placed in a control brooder (room temperature) for 3 h and were bled 1 week prior to and at 1, 2, 4, 6 and 20 h post‐treatment. Treatment had no effect on levels of either constitutive HSP70 or inducible HSP90, but both HSPs decreased with time relative to baseline, suggesting a possible effect of handling stress and/or circadian variation.     

The Effect of Manganese-induced Cytotoxicity on mRNA Expressions of HSP27, HSP40, HSP60, HSP70 and HSP90 in Chicken Spleen Lymphocytes in Vitro

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl₂ (2?×?10^?4, 4?×?10^?4, 6?×?10^?4, 8?×?10^?4, 10?×?10^?4, and 12?×?10^?4 mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.     

Expression pattern of bta-mir-2898 miRNA and their correlation with heat shock proteins during summer heat stress among native vs crossbred cattle

Earlier studies identified the role of bta-mir-2898 in bovine. Our earlier study identified that, bta-mir-2898 can be over expressed in crossbred cattle during heat stress. Nevertheless the differential expression of bta-mir-2898 among native vs crossbred cattle during summer stress along with it's correlation with different heat shock proteins (HSPs) is not yet studied. In the present context, we studied the differential expression of bta-mir-2898 among Frieswal (Bos indicus x Bos taurus) and Sahiwal (Bos indicus) breeds of cattle during a range of environmental air temperatures and further investigated the correlation of bta-mir-2898 with different HSPs (HSP70, HSP90, HSP60. HSF, HSPB8 and HSP27). It was observed that, at peak air temperature the relative miRNA expression level (p < 0.05) of bta-mir-2898 was 3.4 ± 0.41 and 0.79 ± 0.22 among Frieswal and Sahiwal, respectively. We also observed significant levels (p < 0.05) of mRNA abundance of HSP70, HSP90, HSPB8 and HSP27 among the breeds. In all the cases Sahiwal found to exhibited higher level of HSPs in comparison to Frieswal. Studies revealed that the expression profile of bta-mir-2898 was negatively correlated with the expression of all the HSPs during thermal stress in post anti-mir2898 treated PBMC invitro cultured model originated from both Frieswal and Sahiwal cattle breeds. However, significantly (p < 0.05) higher negative correlations were observed between bta-mir-2898 and HSP70, HSP60 and HSPB8. Present findings highlighted the preliminary role of overexpressed bta-mir-2898 in cattle during thermal stress and its impact on different heat shock proteins.     

The 60-kDa Heat Shock Protein (HSP60) of the Sea Anemone <Emphasis Type="BoldItalic">Anemonia viridis:</Emphasis> A Potential Early Warning System for Environmental Changes

Expression of heat shock proteins (HSPs) is often correlated with adaptation to environmental stress. We examined the role of HSP60 (60 kDa) in acclimatization to thermal stress in the sea anemone Anemonia viridis. Using monoclonal antibodies, we identified HSP60 in sea anemones for the first time, and showed that its expression varied with changes in seawater temperature (SWT). Anemonia viridis displayed high levels of HSP60 when extreme temperatures prevailed in stressful habitats such as tidal pools. Specimens sampled from different temperature layers in the same tidal pool differed in their levels of HSP60. Specimens from subtidal zones exhibited a seasonal pattern of expression of HSP60, according to the seasonal SWT. The level of HSP60 was significantly higher in the summer (SWT, 31°C) than in other seasons throughout the year. This study suggests the use of HSP60 expression as a tool for stress detection in marine invertebrates. Received September 25, 2000; accepted February 26, 2001.