Three heat shock proteins are essential for rotifer thermotolerance

Heat shock proteins (HSPs) are important molecules in the stress response of organisms from prokaryotes to mammals, and thus may be useful biomarkers for environmental stress. Here we characterize the functional roles of genes belonging to four distinct families of HSPs (hsp40, hsp60, hsp70, and hsp90) in the monogonont rotifer Brachionus manjavacas. Because B. manjavacas inhabits ponds of varying thermal regimes, including ephemeral ponds that may experience temperature fluctuations, HSP-mediated thermotolerance likely is important to its survival and adaptation. Using interference RNA (RNAi), we provide the first conclusive evidence that HSPs are required for rotifer survival following heat stress. Effective RNAi-mediated suppression of all hsp genes except hsp90 was verified via quantitative PCR. Hsp40, hsp60, and hsp70 are required for rotifer thermotolerance (P < 0.05); however, our data do not indicate hsp90 is essential. Quantitative PCR further revealed immediate up-regulation of hsp40 mRNA following heat stress. Additionally, we demonstrated expression of hsp40 mRNA in multiple tissues using fluorescent in situ hybridization. Our characterization of mRNA expression and functional roles for four distinct hsp genes provides a baseline for molecular-level comparisons of the stress response of rotifers with other taxonomic groups, and the technique for in-depth studies of the role of specific genes in rotifer stress responses. Considering the potential for ambient temperatures to impact species survival, competitive interactions, and body size of individuals, thermotolerance may be an important influence on zooplankton community structure.     

Changes in Expression of Manganese Superoxide Dismutase,Copper and Zinc Superoxide Dismutase and Catalase in Brachionus calyciflorus during the Aging Process

Rotifers are useful model organisms for aging research, owing to their small body size (0.1–1 mm), short lifespan (6–14 days) and the relative easy in which aging and senescence phenotypes can be measured. Recent studies have shown that antioxidants can extend the lifespan of rotifers. In this paper, we analyzed changes in the mRNA expression level of genes encoding the antioxidants manganese superoxide dismutase (MnSOD), copper and zinc SOD (CuZnSOD) and catalase (CAT) during rotifer aging to clarify the function of these enzymes in this process. We also investigated the effects of common life-prolonging methods [dietary restriction (DR) and resveratrol] on the mRNA expression level of these genes. The results showed that the mRNA expression level of MnSOD decreased with aging, whereas that of CuZnSOD increased. The mRNA expression of CAT did not change significantly. This suggests that the ability to eliminate reactive oxygen species (ROS) in the mitochondria reduces with aging, thus aggravating the damaging effect of ROS on the mitochondria. DR significantly increased the mRNA expression level of MnSOD, CuZnSOD and CAT, which might explain why DR is able to extend rotifer lifespan. Although resveratrol also increased the mRNA expression level of MnSOD, it had significant inhibitory effects on the mRNA expression of CuZnSOD and CAT. In short, mRNA expression levels of CAT, MnSOD and CuZnSOD are likely to reflect the ability of mitochondria to eliminate ROS and delay the aging process.     

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA ⁺ background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA⁺ background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA ⁻ background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA ⁺ background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.     

Identifying evolutionarily conserved genes in the dietary restriction response using bioinformatics and subsequent testing in Caenorhabditis elegans

Dietary restriction (DR) increases life span, health span and resistance to stress in a wide range of organisms. Work from a large number of laboratories has revealed evolutionarily conserved mechanisms that mediate the DR response. Here, we analyzed the genome-wide gene expression profiles of Caenorhabditis elegans under DR versus ad libitum conditions. Using the Ortho2ExpressMatrix tool, we searched for C. elegans orthologs of mouse genes that have been shown to be differentially expressed under DR conditions in nearly 600 experiments. Based on our bioinformatic approaches, we obtained 189 DR-responsive genes, and 45 of these are highly conserved from worm to man. Subsequent testing of sixteen genes that are up-regulated under DR identified eight genes that abolish the DR-induced resistance to heat stress in C. elegans. Further analyses revealed that fkb-4, dod-22 and ikb-1 genes also abolish increased life span in response to DR. The identified genes that are necessary for the DR response are sensitive to certain stress signals such as metabolic perturbances (dod-22, fkb-4 and nhr-85), DNA damage (ikb-1), heat shock (hsp-12.6) and cancer-like overgrowth (prk-2 and tsp-15). We propose that most of the DR-responsive genes identified are components of the recently discovered cellular surveillance-activated detoxification and defenses pathway, which is, among others, important for the survival of organisms in times of food deprivation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0363-5) contains supplementary material, which is available to authorized users.     

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments.     

Heat shock proteins mediate trade‐offs between early‐life reproduction and late survival in Drosophila melanogaster

Ageing and the resulting increased likelihood mortality are the inescapable fate of organisms because selection pressures on genes that exert their function late in life is weak, promoting the evolution of genes that enhance early‐life reproductive performance at the same time as sacrificing late survival. Heat shock proteins (HSP) are known to buffer various environmental stresses and are also involved in protein homeostasis and longevity. The characteristics of genes for HSPs (hsp) imply that they affect various life‐history traits, which in turn affect longevity; however, little is known about the effects of hsp genes on life‐history traits and their interaction with longevity. In the present study, the effects of hsp genes on multiple fitness traits, such as locomotor activity, total fecundity, early fecundity and survival time, are investigated in Drosophila melanogaster Meigen using RNA interference (RNAi). In egg‐laying females, RNAi knockdown of six hsp genes (hsp22, hsp23, hsp67Ba, hsp67Bb, hsp67Bc and hsp27‐like) does not shorten survival but rather increases it. Knockdown of five of those genes on an individual basis reduces early‐life reproduction, suggesting that several hsp genes mediate the trade‐off between early reproduction and late survival. The data indicate a positive effect of hsp genes on early reproduction and also negative effects on survival time, supporting the antagonistic pleiotropic effects predicted by the optimality theory of ageing.     

Gene expression during the intra-puparial stage of Chrysomya megacephala: Implications for postmortem interval estimation

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most predominant calliphorid species which arrives and colonizes a cadaver first in its native range of the Australian and Oriental regions, and the intra-puparial stage of this species accounts for about half of its immature stage. Therefore, establishing a reliable aging method of the intra-puparial stage of C. megacephala is very important to accurately estimate the minimum postmortem interval (PMI_min). In this study, actin was used as an internal reference gene to study the expression of three genes, ecdysone receptor gene ecr, white-eye gene white, and heat shock protein gene hsp70, at different time points during intra-puparial development of C. megacephala. Quantification through real-time PCR revealed that these genes can be used to age the intra-puparial period of C. megacephala as they exhibit regular changes and gene expression level was temperature-dependent. The overall gene expression profile of ecr showed a downward trend throughout the intra-puparial stage. White expression increased steadily until it peaked when intra-puparial development reached 45%, and the expression began to decrease when the intra-puparial development reached 55%. hsp70 was first down-regulated at 0–15% of intra-puparial development, and then slightly up-regulated at 15–40%, and finally down-regulated again until the end of intra-puparial development. This study provides molecular indicators of age during the intra-puparial stage of C. megacephala, and combining gene expression with the morphological methods can lead to more accurate estimation for PMI_min.     

Evolutionary implications of a complex pattern of DNA sequence homology extending far upstream of the hsp70 genes at loci 87A7 and 87C1 in Drosophila melanogaster

Genes coding for the major 70,000 M_r heat shock protein (hsp70) are found at two loci, 87A7 and 87C1, in Drosophila melanogaster. At 87A7 they are present as two genes in diverging orientation, whilst at 87C1 two tandemly repeated distal copies are separated from a single copy in divergent orientation by about 40,000 bases of DNA. Within this 40,000 bases are found the αβ heat-induced genes, interspersed with γ elements. In this paper we report the isolation and characterization of the proximal hsp70 gene from locus 87C1. The DNA sequence upstream from this gene shows greater than 98% homology with that of αγ, suggesting that the γ element interspersed with αβ sequences originated from this position. In addition, we present the DNA sequence between the two genes in a cloned DNA segment from 87A7, and compare the sequence with those from 87C1. We find a complex pattern of nucleotide sequence homology extending far upstream of the hsp70 genes at the two loci. The evolution of the present arrangement at these two loci is discussed.     

Effects of 20-OH-ecdysone on Drosophila cells: Regulation of endogenous and transfected genes

Drosophila cell lines respond to physiological doses of 20-OH-ecdysone by entering mitotic arrest and differentiating morphologically. The cells also exhibit changes in gene expression. Several enzyme activities are induced, and the synthesis of cytoplasmic actin and of the four small heat-shock proteins (hsp) is initiated. Hybrid genes, containing the 5′ region of Drosophila heat-shock protein genes ligated to the herpes simplex virus thymidine kinase gene (tk), have been transfected into cells of the Drosophila cell line S3. Constructions containing sequences upstream from hsp 70, or from any of the small hsp genes, show heat-inducible tk expression. Ecdysterone-inducible tk expression is seen only in transfections with small hsp-tk hybrid genes. This transient expression system can be used as an assay for function to define regions of DNA, flanking the coding region of inducible genes, which are necessary for normal gene expression and gene regulation in cultured cells.     

Identification and characterization of novel ER-based hsp90 gene in the red flour beetle,Tribolium castaneum

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0487-y) contains supplementary material, which is available to authorized users.     

The effect of neuronal expression of heat shock proteins 26 and 27 on lifespan, neurodegeneration, and apoptosis in Drosophila

Heat shock proteins (Hsps) are chaperones thought to increase lifespan, enhance stress resistance, and prevent apoptosis and neurodegenerative diseases. Our previous study reported that ubiquitous expression of hsp26 or hsp27 extended Drosophila lifespan. The effect of neuronal expression of hsp26 and hsp27 in Drosophila on the above-mentioned functions has not yet been investigated. Here, we show that neuronal expression of hsp26 or hsp27 improved lifespan and increased resistance to oxidative stress. However, only neuronal expression of hsp27 ameliorated Parkinsonism climbing disorder and attenuated mild polyglutamine-induced toxicity. Additionally, neuronal expression of hsp27 specifically partially rescued hid-induced lethality, but was not able to rescue reaper/grim-induced lethality. However, unlike hsp27, neuronal expression of hsp26 did not rescue hid-induced or reaper/grim-induced lethality. In summary, we demonstrate the functional similarities and differences of neuronal expression of hsp26 and hsp27 in adult Drosophila.     

Molecular Characterization of Mn-superoxide Dismutase and Gene Expression Studies in Dietary Restricted <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Rotifers

Superoxide dismutases (SODs) promote a conversion of harmful reactive oxygen species (ROS) to relatively moderate forms, resulting in the extension of lifespan in the nematode Caenorhabditis elegans under caloric restriction. The lifespan of the rotifer Brachionus plicatilis is also markedly extended by caloric restriction. We, therefore, cloned cDNA encoding SOD activated with Mn (Mn SOD) from B. plicatilis and examined its expression pattern in rotifers raised with energy restricted diet. The full length deduced amino acid sequence of the rotifer Mn SOD showed 61% identity with the C. elegans ortholog. Four amino acid residues that are essential to the binding of this enzyme to Mn were conserved in the rotifer Mn SOD. Subsequently we examined the mRNA expression patterns of Mn SOD using highly sensitive quantitative real-time PCR for various rotifer populations that are likely to differ in their lifespans in experiments on calorie restricted diets. The accumulated mRNA levels of Mn SOD were found to increase in supposedly long-lived rotifers. These results suggest that Mn SOD is possibly related to the aging of B. plicatilis.     

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.