Cytoplasmic aggregation of mutant FUS causes multistep RNA splicing perturbations in the course of motor neuron pathology.

Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.

Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis.

Syndromes of hybrid dysgenesis (HD) have been critical for our understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a "neutral" cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, we performed a comparative analysis of the landscape of ovarian small RNAs in strains that vary in their resistance to HD mediated sterility. We demonstrate that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, we have witnessed a cytotype shift from neutral (N) to susceptible (M) in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, our data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD.

Evolution and dynamics of small RNA response to a retroelement invasion in Drosophila.

Although small RNAs efficiently control transposition activity of most transposons in the host genome, such an immune system is not always applicable against a new transposon's invasions. Here, we explored a possibility to introduce potentially mobile copy of the Penelope retroelement previously implicated in hybrid dysgenesis syndrome in Drosophila virilis into the genomes of two distant Drosophila species. The consequences of such introduction were monitored at different phases after experimental colonization as well as in D. virilis species, which is apparently in the process of ongoing Penelope invasion. We investigated the expression of Penelope and biogenesis of Penelope-derived small RNAs in D. virilis and D. melanogaster strains originally lacking active copies of this element after experimental Penelope invasion. These strains were transformed by constructs containing intact Penelope copies. We show that immediately after transformation, which imitates the first stage of retroelement invasion, Penelope undergoes transposition predominantly in somatic tissues, and may produce siRNAs that are apparently unable to completely silence its activity. However, at the later stages of colonization Penelope copies may jump into one of the piRNA-clusters, which results in production of homologous piRNAs that are maternally deposited and can silence euchromatic transcriptionally active copies of Penelope in trans and, hence, prevent further amplification of the invader in the host genome. Intact Penelope copies and different classes of Penelope-derived small RNAs were found in most geographical strains of D. virilis collected throughout the world. Importantly, all strains of this species containing full-length Penelope tested do not produce gonadal sterility in dysgenic crosses and, hence, exhibit neutral cytotype. To understand whether RNA interference mechanism able to target Penelope operates in related species of the virilis group, we correlated the presence of full-length and potentially active Penelope with the occurrence of piRNAs homologous to this transposable element in the ovaries of species comprising the group. It was demonstrated that Penelope-derived piRNAs are present in all virilis group species containing full-length but transcriptionally silent copies of this element that probably represent the remnants of its previous invasions taking place in the course of the virilis species divergent evolution.

Exogenous Hsp70 exerts neuroprotective effects in peripheral nerve rupture model.

Hsp70 is the main molecular chaperone responsible for cellular proteostasis under normal conditions and for restoring the conformation or utilization of proteins damaged by stress. Increased expression of endogenous Hsp70 or administration of exogenous Hsp70 is known to exert neuroprotective effects in models of many neurodegenerative diseases. In this study, we have investigated the effect of exogenous Hsp70 on recovery from peripheral nerve injury in a model of sciatic nerve transection in rats. It was shown that recombinant Hsp70 after being added to the conduit connecting the ends of the nerve at the site of its extended severance, migrates along the nerve into the spinal ganglion and is retained there at least three days. In animals with the addition of recombinant Hsp70 to the conduit, a decrease in apoptosis in the spinal ganglion cells after nerve rupture, an increase in the level of PTEN-induced kinase 1 (PINK1), an increase in markers of nerve tissue regeneration and a decrease in functional deficit were observed compared to control animals. The obtained data indicate the possibility of using recombinant Hsp70 preparations to accelerate the recovery of patients after neurotrauma.

Cell-Permeable HSP70 Protects Neurons and Astrocytes Against Cell Death in the Rotenone-Induced and Familial Models of Parkinson's Disease.

Heat shock protein 70 (HSP70) is activated under stress response. Its involvement in cell protection, including energy metabolism and quality control makes it a promising pharmacological target. A strategy to increase HSP70 levels inside the cells is the application of recombinant HSP70. However, cell permeability and functionality of these exogenously applied proteins inside the cells is still disputable. Here, using fluorescence- labeled HSP70, we have studied permeability and distribution of HSP70 inside primary neurons and astrocytes, and how exogenous HSP70 changes mitochondrial metabolism and mitophagy. We have found that exogenous recombinant HSP70 can penetrate the neurons and astrocytes and distributes in mitochondria, lysosomes and in lesser degree in the endoplasmic reticulum. HSP70 increases mitochondrial membrane potential in control neurons and astrocytes, and in fibroblasts of patients with familial Parkinson´s disease (PD) with PINK1 and LRRK2 mutations. Increased mitochondrial membrane potential was associated with higher mitochondrial ROS production and activation of mitophagy. Importantly, preincubation of the cells with HSP70 protected neurons and astrocytes against cell death in a toxic model of PD induced by rotenone, and in the PINK1 and LRRK2 PD human fibroblasts. Thus, exogenous recombinant HSP70 is cell permeable, and acts as endogenous HSP70 protecting cells in the case of toxic model and familial forms of Parkinson's Disease.

Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome.

BACKGROUND: Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats. RESULTS: Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed. CONCLUSION: Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

Retroelements containing introns in diverse invertebrate taxa.

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.

The Role of Hsp70 in Adaptation to Adverse Conditions and Its Possible Medical Application.

In the present era of global warming and dramatically increased environmental pollution posing a threat to animal life, the understanding and manipulation of organisms' resources of stress tolerance is apparently a question of survival. Heat stress and other forms of stressful factors induce a highly organized response of organisms at the cellular level where heat shock proteins (Hsps) and in particular Hsp70 family of chaperones are among the major players in the protection from the environmental challenge. The present review article summarizes the peculiarities of the Hsp70 family of proteins protective functions being a result of many millions of years of adaptive evolution. It discusses the molecular structure and specific details of hsp70 gene regulation in various organisms, living in diverse climatic zones, with a special emphasis on the protective role of Hsp70 in adverse conditions of the environment. The review discusses the molecular mechanisms underlying Hsp70-specific properties that emerged in the course of adaptation to harsh environmental conditions. This review also includes the data on the anti-inflammatory role of Hsp70 and the involvement of endogenous and recombinant Hsp70 (recHsp70) in proteostatic machinery in various pathologies including neurodegenerative ones such as Alzheimer's and Parkinson's diseases in rodent model organisms and humans in vivo and in vitro. Specifically, the role of Hsp70 as an indicator of disease type and severity and the use of recHsp70 in several pathologies are discussed. The review discusses different roles exhibited by Hsp70 in various diseases including the dual and sometimes antagonistic role of this chaperone in various forms of cancer and viral infection including the SARS-Cov-2 case. Since Hsp70 apparently plays an important role in many diseases and pathologies and has significant therapeutic potential there is a dire need to develop cheap recombinant Hsp70 production and further investigate the interaction of externally supplied and endogenous Hsp70 in chaperonotherapy.

The role of molecular chaperones in the mechanisms of epileptogenesis.

Epilepsy is a group of neurological diseases which requires significant economic costs for the treatment and care of patients. The central point of epileptogenesis stems from the failure of synaptic signal transmission mechanisms, leading to excessive synchronous excitation of neurons and characteristic epileptic electroencephalogram activity, in typical cases being manifested as seizures and loss of consciousness. The causes of epilepsy are extremely diverse, which is one of the reasons for the complexity of selecting a treatment regimen for each individual case and the high frequency of pharmacoresistant cases. Therefore, the search for new drugs and methods of epilepsy treatment requires an advanced study of the molecular mechanisms of epileptogenesis. In this regard, the investigation of molecular chaperones as potential mediators of epileptogenesis seems promising because the chaperones are involved in the processing and regulation of the activity of many key proteins directly responsible for the generation of abnormal neuronal excitation in epilepsy. In this review, we try to systematize current data on the role of molecular chaperones in epileptogenesis and discuss the prospects for the use of chemical modulators of various chaperone groups' activity as promising antiepileptic drugs.

Small RNA-based silencing strategies for transposons in the process of invading Drosophila species.

Colonization of a host by an active transposon can increase mutation rates or cause sterility, a phenotype termed hybrid dysgenesis. As an example, intercrosses of certain Drosophila virilis strains can produce dysgenic progeny. The Penelope element is present only in a subset of laboratory strains and has been implicated as a causative agent of the dysgenic phenotype. We have also introduced Penelope into Drosophila melanogaster, which are otherwise naive to the element. We have taken advantage of these natural and experimentally induced colonization processes to probe the evolution of small RNA pathways in response to transposon challenge. In both species, Penelope was predominantly targeted by endo-small-interfering RNAs (siRNAs) rather than by piwi-interacting RNAs (piRNAs). Although we do observe correlations between Penelope transcription and dysgenesis, we could not correlate differences in maternally deposited Penelope piRNAs with the sterility of progeny. Instead, we found that strains that produced dysgenic progeny differed in their production of piRNAs from clusters in subtelomeric regions, possibly indicating that changes in the overall piRNA repertoire underlie dysgenesis. Considered together, our data reveal unexpected plasticity in small RNA pathways in germ cells, both in the character of their responses to invading transposons and in the piRNA clusters that define their ability to respond to mobile elements.

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster.

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

Paramutation-like Epigenetic Conversion by piRNA at the Telomere of Drosophila virilis.

First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation.

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. RESULTS: Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. CONCLUSIONS: The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process.

Role of a Heat Shock Transcription Factor and the Major Heat Shock Protein Hsp70 in Memory Formation and Neuroprotection.

Heat shock proteins (Hsps) represent the most evolutionarily ancient, conserved, and universal system for protecting cells and the whole body from various types of stress. Among Hsps, the group of proteins with a molecular weight of 70 kDa (Hsp70) plays a particularly important role. These proteins are molecular chaperones that restore the native conformation of partially denatured proteins after exposure to proteotoxic forms of stress and are critical for the folding and intracellular trafficking of de novo synthesized proteins under normal conditions. Hsp70s are expressed at high levels in the central nervous system (CNS) of various animals and protect neurons from various types of stress, including heat shock, hypoxia, and toxins. Numerous molecular and behavioral studies have indicated that Hsp70s expressed in the CNS are important for memory formation. These proteins contribute to the folding and transport of synaptic proteins, modulate signaling cascades associated with synaptic activation, and participate in mechanisms of neurotransmitter release. In addition, HSF1, a transcription factor that is activated under stress conditions and mediates Hsps transcription, is also involved in the transcription of genes encoding many synaptic proteins, whose levels are increased in neurons under stress and during memory formation. Thus, stress activates the molecular mechanisms of memory formation, thereby allowing animals to better remember and later avoid potentially dangerous stimuli. Finally, Hsp70 has significant protective potential in neurodegenerative diseases. Increasing the level of endogenous Hsp70 synthesis or injecting exogenous Hsp70 reduces neurodegeneration, stimulates neurogenesis, and restores memory in animal models of ischemia and Alzheimer's disease. These findings allow us to consider recombinant Hsp70 and/or Hsp70 pharmacological inducers as potential drugs for use in the treatment of ischemic injury and neurodegenerative disorders.

The Role of p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy.

The search for effective neuroprotective agents for the treatment of neurotrauma has always been of great interest to researchers around the world. Extracellular heat shock protein 70 (eHsp70) is considered a promising agent to study, as it has been demonstrated to exert a significant neuroprotective activity against various neurodegenerative diseases. We showed that eHsp70 can penetrate neurons and glial cells when added to the incubation medium, and can accumulate in the nuclei of neurons and satellite glial cells after axotomy. eHsp70 reduces apoptosis and necrosis of the glial cells, but not the neurons. At the same time, co-localization of eHsp70 with p53 protein, one of the key regulators of apoptosis, was noted. eHsp70 reduces the level of the p53 protein apoptosis promoter both in glial cells and in the nuclei and cytoplasm of neurons, which indicates its neuroprotective effect. The ability of eHsp70 to reverse the proapoptotic effect of the p53 activator WR1065 may indicate its ability to regulate p53 activity or its proteosome-dependent degradation.

Transcriptome of the Krushinsky-Molodkina Audiogenic Rat Strain and Identification of Possible Audiogenic Epilepsy-Associated Genes.

Audiogenic epilepsy (AE), inherent to several rodent strains is widely studied as a model of generalized convulsive epilepsy. The molecular mechanisms that determine the manifestation of AE are not well understood. In the present work, we compared transcriptomes from the corpora quadrigemina in the midbrain zone, which are crucial for AE development, to identify genes associated with the AE phenotype. Three rat strains without sound exposure were compared: Krushinsky-Molodkina (KM) strain (100% AE-prone); Wistar outbred rat strain (non-AE prone) and "0" strain (partially AE-prone), selected from F2 KM × Wistar hybrids for their lack of AE. The findings showed that the KM strain gene expression profile exhibited a number of characteristics that differed from those of the Wistar and "0" strain profiles. In particular, the KM rats showed increased expression of a number of genes involved in the positive regulation of the MAPK signaling cascade and genes involved in the positive regulation of apoptotic processes. Another characteristic of the KM strain which differed from that of the Wistar and "0" rats was a multi-fold increase in the expression level of the Ttr gene and a significant decrease in the expression of the Msh3 gene. Decreased expression of a number of oxidative phosphorylation-related genes and a few other genes was also identified in the KM strain. Our data confirm the complex multigenic nature of AE inheritance in rodents. A comparison with data obtained from other independently selected AE-prone rodent strains suggests some common causes for the formation of the audiogenic phenotype.

Evolutionary Dynamics of the Pericentromeric Heterochromatin in Drosophila virilis and Related Species.

Pericentromeric heterochromatin in Drosophila generally consists of repetitive DNA, forming the environment associated with gene silencing. Despite the expanding knowledge of the impact of transposable elements (TEs) on the host genome, little is known about the evolution of pericentromeric heterochromatin, its structural composition, and age. During the evolution of the Drosophilidae, hundreds of genes have become embedded within pericentromeric regions yet retained activity. We investigated a pericentromeric heterochromatin fragment found in D. virilis and related species, describing the evolution of genes in this region and the age of TE invasion. Regardless of the heterochromatic environment, the amino acid composition of the genes is under purifying selection. However, the selective pressure affects parts of genes in varying degrees, resulting in expansion of gene introns due to TEs invasion. According to the divergence of TEs, the pericentromeric heterochromatin of the species of virilis group began to form more than 20 million years ago by invasions of retroelements, miniature inverted repeat transposable elements (MITEs), and Helitrons. Importantly, invasions into the heterochromatin continue to occur by TEs that fall under the scope of piRNA silencing. Thus, the pericentromeric heterochromatin, in spite of its ability to induce silencing, has the means for being dynamic, incorporating the regions of active transcription.

Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins.

Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. Here, we find that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, we demonstrate that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, we show that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. We propose that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin.

The molecular chaperone Hsp70 from the thermotolerant Diptera species differs from the Drosophila paralog in its thermostability and higher refolding capacity at extreme temperatures.

Previously, we demonstrated that species of the Stratiomyidae family exhibit higher tolerance to thermal stress in comparison with that of many representatives of Diptera, including Drosophila species. We hypothesized that species of this group inherited the specific structures of their chaperones from an ancestor of the Stratiomyidae family, and this enabled the descendants to colonize various extreme habitats. To explore this possibility, we cloned and expressed in Escherichia coli copies of the Hsp70 genes from Stratiomys singularior, a typical eurythermal species, and Drosophila melanogaster, for comparison. To investigate the thermal sensitivity of the chaperone function of the inducible 70-kDa heat shock proteins from these species, we used an in vitro refolding luciferase assay. We demonstrated that under conditions of elevated temperature, S. singularior Hsp70 exhibited higher reactivation activity in comparison with D. melanogaster Hsp70 and even human Hsp70. Similarly, S. singularior Hsp70 was significantly more thermostable and showed in vitro refolding activity after preheatment at higher temperatures than D. melanogaster paralog. Thermally induced unfolding experiments using differential scanning calorimetry indicated that Hsp70 from both Diptera species is formed by two domains with different thermal stabilities and that the ATP-binding domain of S. singularior is stable at temperatures 4 degrees higher than that of the D. melanogaster paralog. To the best of our knowledge, this study represents the first report that provides direct experimental data indicating that the evolutionary history of a species may result in adaptive changes in the structures of chaperones to enable them to elicit protective functions at extreme environments.

A partial genome assembly of the miniature parasitoid wasp, Megaphragma amalphitanum.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.