A transcriptome-wide assessment of differentially expressed genes among two highly divergent,yet sympatric,lineages of the freshwater Atyid shrimp, <Emphasis Type="Italic">Paratya australiensis</Emphasis>

The current study evaluated the possible toxic effects of the water-soluble fraction of crude oil on the general cellular stress-response mechanisms of two dominant representatives of Lake Baikal’s littoral community, the endemic amphipod species Eulimnogammarus verrucosus and E. cyaneus. The acute toxicity effects on the cellular stress-response mechanisms of amphipods were studied in the laboratory by exposing amphipods in water from Lake Baikal to addition of a water-soluble fraction of crude oil at concentrations considered safe for the aquatic environment. The present study found that even short-term exposure to a water-soluble fraction of crude oil at concentration of 50 µg/L, established as the threshold limit for fishery and aquaculture water reservoirs in the Russian Federation, directly affected the general stress-response markers HSP70 and lipid peroxidation and significantly changed the activity of antioxidant enzymes in both studied species. This result confirms the high sensitivity of Baikal endemics to crude oil. Thus, it also indicates that established standards and threshold limit values of oil concentrations estimated for ecological monitoring of general water reservoirs cannot be applied directly to the unique Lake Baikal ecosystem.     

Heat shock proteins in the mechanisms of stress adaptation in Baikal amphipods and palearctic <Emphasis Type="Italic">Gammarus lacustris</Emphasis> Sars. I. HSP70 family

The participation of HSP70 family proteins in the mechanisms of stress adaptation under thermal and toxic stress in Baikal endemic and palearctic organisms was investigated. The four Lake Baikal endemic species Gmelinoides fasciatus (Stebb.), Eulimnogammarus cyaneus (Dyb.), E. vittatus (Dyb.), Ommatogammarus fl avus (Dyb.) were compared with the palearctic fauna representative Gammarus lacustris Sars. The type of synthesis of HSP70 family heat shock proteins under thermal (exposure at a temperature of 20, 25, and 30°C) and toxic (exposure in CdCl₂ solutions with the concentrations 50, 10, 5, 0.5, and 0.05 mg/l) stress was determined. All species under investigation were shown to have a common trend towards HSP70 content increase; therewith some species-specific characteristics of the type of synthesis of the protein under investigation were observed. The conclusion has been made on HSP70 participation in the mechanisms of thermal and toxic resistance in the studied amphipod species.     

Heat shock proteins in the mechanisms of stress adaptation in Baikal amphipods and Palaearctic <Emphasis Type="Italic">Gammarus lacustris</Emphasis> Sars II. Small HSP family

The role of low-molecular-weight (small) heat shock proteins (sHSPs) in the mechanisms of thermal and toxic resistance in fresh-water organisms was investigated. The four endemic species of Lake Baikal: Gmelinoides fasciatus (Stebb.), Eulimnogammarus cyaneus (Dyb.), E. vittatus (Dyb.), and Ommatogammarus flavus (Dyb.), and Gammarus lacustris Sars, a representative of the Palaearctic fauna, were studied. The effect of the temperature factor was evaluated by exposure of amphipods to temperatures of 20, 25, and 30°C, and the effect of the toxic factor, by exposure to cadmium chloride solutions with concentrations of 50, 10, 5, 0.5, and 0.05 mg/l. All the species showed a common trend of increasing content of sHSPs; species-specific features were observed in the synthesis of the proteins. It was concluded that sHSPs participate in the mechanisms of thermal and toxic resistance in the investigated amphipod species.     

Thermal stress defense in freshwater amphipods from contrasting habitats with emphasis on small heat shock proteins (sHSPs)

This study comparatively evaluated small heat shock proteins (sHSP) (related to α-crystallin) and antioxidant enzymes such as peroxidase (POD), catalase (CAT), glutathione S-transferase (GST) as anti-thermal stress response in two contrasting freshwater amphipods, the stenoecious Baikalean endemic Eulimnogammarus cyaneus and the Palearctic Gammarus lacustris.The thermal stress modulated the activity of antioxidant enzymes as well as the sHSP synthesis in both species. In both species, only the declining POD activity showed a clear dependency on exposure time.The most expressed response to elevated temperatures has been the activation of sHSP synthesis, with clear differences in the patterns: in G. lacustris, sHSP concentrations peaked after 12 h with a subsequent decline, while they increased steadily in E. cyaneus. Hence, the stenoecious species did not acclimate to the thermal stress within the given exposure time as the euryecious did.     

Use of surface-enhanced laser desorption ionization-time-of-flight to identify heat shock protein 70 isoforms in closely related species of the virilis group of Drosophila

The 70-kDa heat shock protein (Hsp) family in all Drosophila species includes 2 environmentally inducible family members, Hsp70 and Hsp68. Two-dimensional gel electrophoresis revealed an unusual pattern of heat shock-inducible proteins in the species of the virilis group. Trypsin fingerprinting and microsequencing of tryptic peptides using ProteinChip Array technology identified the major isoelectric variants of Hsp70 family, including Hsp68 isoforms that differ in both molecular mass and isoelectric point from those in Drosophila melanogaster. The peculiar electrophoretic mobility is consistent with the deduced amino acid sequence of corresponding hsp genes from the species of the virilis group.     

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.     

Cloning and expression of heat shock protein genes in two whitefly species in response to thermal stress

Two whitefly species, Trialeurodes vaporariorum and Bemisia tabaci biotype B were shown to have different temperature tolerance and seasonal dynamics. To determine whether this variation in thermal tolerance is related to different expression patterns of heat shock protein (hsp) genes during temperature stress, we obtained complete cDNA sequences for hsp90, hsp70 and hsp20, and analysed their expression profiles across temperature gradients by real‐time quantitative polymerase chain reaction (PCR). Six full‐length cDNAs were cloned and sequenced from these two species. The full‐length cDNAs of hsp90s contain 2166 and 2157 bp open‐reading frames (ORF) which encode proteins with calculated molecular weights of 83 013 and 82 857 Da in T. vaporariorum and B. tabaci, respectively. The 1947 and 1959 bp ORFs of whitefly hsp70s comprise 649 and 653 amino acids with the calculated masses of 70 885 and 71 008 Da in T. vaporariorum and B. tabaci, respectively. Both complete cDNAs of hsp20 of T. vaporariorum and B. tabaci contain 585 bp ORFs and deduced amino acid sequences had molecular weights of 21 559 and 21 539 Da, respectively. The hsp expression profile results showed that temperatures for onset (T_on) or maximal (T_max) induction of hsp expression in T. vaporariorum were generally 2–6°C lower than those in B. tabaci. These results suggest that the T_on (or T_max) of hsps can represent the differences in temperature tolerance of these two whitefly species, and may be used to determine their natural geographical distribution and natural population seasonal dynamics. Significant upregulation of most hsps were observed when temperature stress was lifted, except that hsp70 and hsp20 of B. tabaci did not respond to the cold stress, indicating that response to heat and cold stress may have a different genetic and physiological basis in two whitefly species. These results highlight the importance of understanding the complexity of the heat shock response across multiple isoforms while attempting to link them to whole‐organism traits such as thermal tolerance.     

Natural and Genetic Engineering of the Heat-Shock Protein Hsp70 in Drosophila melanogaster: Consequences for Thermotolerance

SYNOPSIS. Larvae of the fruit fly, Drosophila melanogaster,live within necrotic fruit, a challenging environment in whichlarvae can experience severe thermal stress. One response tothermal stress, the expression of heat-shock proteins (Hsps),has evolved distinctively in this species; the gene encodingHsp70 has undergone extensive duplication and accounts for thebulk of Hsps that are expressed upon heat shock. Genetic engineeringof hsp70 copy number is sufficient to affect thermotoleranceat some (but not all) life stages. Increases in Hsp70, moreover,can protect intact larvae against thermal inactivation of theenzyme alcohol dehydrogenase and thermal inhibition of feeding.Deleterious consequences of high levels of Hsp70, however, maylimit further evolutionary proliferation of hsp70 genes. Thesefindings illustrate how the perspectives of integrative andcomparative biology, if applied to even well-studied model organisms,can lead to novel findings.     

Microsporidian Parasites Found in the Hemolymph of Four Baikalian Endemic Amphipods

At present, approximately 187 genera and over 1300 species of Microsporidia have been described, among which almost half infect aquatic species and approximately 50 genera potentially infect aquatic arthropods. Lake Baikal is the deepest and one of the oldest lakes in the world, and it has a rich endemic fauna with a predominance of arthropods. Among the arthropods living in this lake, amphipods (Crustacea) are the most dominant group and are represented by more than 350 endemic species. Baikalian amphipods inhabit almost all depths and all types of substrates. The age and geographical isolation of this group creates excellent opportunities for studying the diversity, evolution and genetics of host-parasite relationships. However, despite more than 150 years of study, data investigating the microsporidia of Lake Baikal remain incomplete. In this study, we used molecular genetic analyses to detect microsporidia in the hemolymph of several endemic species of amphipods from Lake Baikal. We provide the first evidence that microsporidian species belonging to three genera (Microsporidium, Dictyocoela and Nosema) are present in the hemolymph of Baikalian endemic amphipods. In the hemolymph of Eulimnogammarus verrucosus, we detected SSU rDNA of microsporidia belonging to the genus Nozema. In the hemolymph of Pallasea cancellous, we found the DNA of Microsporidium sp. similar to that in other Baikalian endemic amphipods; Dictyocoela sp. was found in the hemolymph of Eulimnogammarus marituji and Acanthogammarus lappaceus longispinus.     

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments

Background  

Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters.     

The role of the heat shock proteins (HSP70 and sHSP) in the thermotolerance of freshwater amphipods from contrasting habitats

The aim of this study was to clarify the significance of HSP70 and sHSP for thermotolerance in freshwater amphipods. We compared four amphipod species from different freshwater habitats and biogeographical regions (Central Europe vs. Lake Baikal). Test individuals were exposed to thermal stress generated by a water temperature of 25 °C. The thermotolerance of the species, determined by median lethal time (LT50), followed in decreasing order by Gmelinoides fasciatus, Echinogammarus berilloni, Gammarus pulex, Eulimnogammarus verrucosus. HSP70 and sHSP base level concentrations for the species were determined at control (i.e. non-stress) conditions. For HSP70, the base levels were positively correlated to the species' thermotolerances. For sHSP, however, only thermotolerant G. fasciatus showed a high level. Thermal stress at 25 °C water temperature caused a deferred onset of HSP70 and sHSP expression followed by a subsequent offset, delineating a unimodal response curve. The time lag to the expression onset of HSP70 was shorter in the thermosensitive species, compared to thermotolerant ones. Conversely, the time span until the maximum level of HSP70 was variable, not showing a dependence on the thermotolerance properties of the species. The peak concentration in G. pulex was distinctly higher than in the other species, whereas E. verrucosus did not develop a well-defined response maximum at all. In sHSP, the temporal pattern of expression was even more variable than in HSP70. However, the thermosensitive species E. verrucosus showed a time lag of expression onset significantly shorter than the other species and thermotolerant G. fasciatus developed the most pronounced response maximum. Basing on these results, the cellular response to thermal stress in amphipods is more consistently reflected by HSP70, compared to sHSP.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

Improved adaptation to heat,cold, and solvent tolerance in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.