Exposure to the Natural Compound Climacostol Induces Cell Damage and Oxidative Stress in the Fruit Fly Drosophila melanogaster.

The ciliate Climacostomum virens produces the metabolite climacostol that displays antimicrobial activity and cytotoxicity on human and rodent tumor cells. Given its potential as a backbone in pharmacological studies, we used the fruit fly Drosophila melanogaster to evaluate how the xenobiotic climacostol affects biological systems in vivo at the organismal level. Food administration with climacostol demonstrated its harmful role during larvae developmental stages but not pupation. The midgut of eclosed larvae showed apoptosis and increased generation of reactive oxygen species (ROS), thus demonstrating gastrointestinal toxicity. Climacostol did not affect enteroendocrine cell proliferation, suggesting moderate damage that does not initiate the repairing program. The fact that climacostol increased brain ROS and inhibited the proliferation of neural cells revealed a systemic (neurotoxic) role of this harmful substance. In this line, we found lower expression of relevant antioxidant enzymes in the larvae and impaired mitochondrial activity. Adult offsprings presented no major alterations in survival and mobility, as well the absence of abnormal phenotypes. However, mitochondrial activity and oviposition behavior was somewhat affected, indicating the chronic toxicity of climacostol, which continues moderately until adult stages. These results revealed for the first time the detrimental role of ingested climacostol in a non-target multicellular organism.

Neuroprotective role of plumbagin on eye damage induced by high-sucrose diet in adult fruit fly Drosophila melanogaster.

The natural compound plumbagin has a wide range of pharmacological and potential therapeutic activities, although its role in neuroretina degeneration is unknown. Here we evaluated the effects of plumbagin on retina homeostasis of the fruit fly Drosophila melanogaster fed with high glucose diet, a model of hyperglycemia-induced eye impairment to study the pathophysiology of diabetic retinopathy at the early stages. To this aim, the visual system of flies orally administered with plumbagin has been analyzed at structural, functional, and molecular/cellular level as for instance neuronal apoptosis/autophagy dysregulation and oxidative stress-related signals. Our results demonstrated that plumbagin ameliorates the visual performance of hyperglycemic flies. Drosophila eye-structure, clearly altered by hyperglycemia, i.e. defects of the pattern of ommatidia, irregular rhabdomeres, vacuoles, damaged mitochondria, and abnormal phototransduction units were rescued, at least in part, by plumbagin. In addition, it reactivated autophagy, decreased the presence of cell death/apoptotic features, and exerted antioxidant effects in the retina. In terms of mechanisms favoring death/survival ratio, Nrf2 signaling activation may be one of the strategies by which plumbagin reduced redox unbalance mainly increasing the levels of glutathione-S-transferase. Likewise, plumbagin may act additively and/or synergistically inhibiting the mitochondrial-endoplasmic reticulum stress and unfolded protein response pathways, which prevented neuronal impairment and eye damage induced by reactive oxygen species. These results provide an avenue for further studies, which may be helpful to develop novel therapeutic candidates and drug targets against eye neurotoxicity by high glucose, a key aspect in retinal complications of diabetes.

RACK1 is evolutionary conserved in satellite stem cell activation and adult skeletal muscle regeneration.

Skeletal muscle growth and regeneration involves the activity of resident adult stem cells, namely satellite cells (SC). Despite numerous mechanisms have been described, different signals are emerging as relevant in SC homeostasis. Here we demonstrated that the Receptor for Activated C-Kinase 1 (RACK1) is important in SC function. RACK1 was expressed transiently in the skeletal muscle of post-natal mice, being abundant in the early phase of muscle growth and almost disappearing in adult mature fibers. The presence of RACK1 in interstitial SC was also detected. After acute injury in muscle of both mouse and the fruit fly Drosophila melanogaster (used as alternative in vivo model) we found that RACK1 accumulated in regenerating fibers while it declined with the progression of repair process. To note, RACK1 also localized in the active SC that populate recovering tissue. The dynamics of RACK1 levels in isolated adult SC of mice, i.e., progressively high during differentiation and low compared to proliferating conditions, and RACK1 silencing indicated that RACK1 promotes both the formation of myotubes and the accretion of nascent myotubes. In Drosophila with depleted RACK1 in all muscle cells or, specifically, in SC lineage we observed a delayed recovery of skeletal muscle after physical damage as well as the low presence of active SC in the wound area. Our results also suggest the coupling of RACK1 to muscle unfolded protein response during SC activation. Collectively, we provided the first evidence that transient levels of the evolutionarily conserved factor RACK1 are critical for adult SC activation and proper skeletal muscle regeneration, favoring the efficient progression of SC from a committed to a fully differentiated state.

Apple Polyphenol Diet Extends Lifespan, Slows down Mitotic Rate and Reduces Morphometric Parameters in Drosophila Melanogaster: A Comparison between Three Different Apple Cultivars.

Plant-derived polyphenols exhibit beneficial effects on physiological and pathological processes, including cancer and neurodegenerative disorders, mainly because of their antioxidant activity. Apples are highly enriched in these compounds, mainly in their peel. The Tuscia Red (TR) apple variety exhibits the peculiar characteristic of depositing high quantities of polyphenols in the pulp, the edible part of the fruit. Since polyphenols, as any natural product, cannot be considered a panacea per se, in this paper, we propose to assess the biological effects of TR flesh extracts, in comparison with two commercial varieties, in a model system, the insect Drosophila melanogaster, largely recognized as a reliable system to test the in vivo effects of natural and synthetic compounds. We performed a comparative, qualitative and quantitative analysis of the polyphenol compositions of the three cultivars and found that TR flesh shows the highest content of polyphenols, and markedly, anthocyanins. Then, we focused on their effects on a panel of physiological, morphometrical, cellular and behavioral phenotypes in wild-type D. melanogaster. We found that all the apple polyphenol extracts showed dose-dependent effects on most of the phenotypes we considered. Remarkably, all the varieties induced a strong relenting of the cell division rate.

Nutraceutical Strategy to Counteract Eye Neurodegeneration and Oxidative Stress in Drosophila melanogaster Fed with High-Sugar Diet.

Aberrant production of reactive oxygen species (ROS) is a common feature of damaged retinal neurons in diabetic retinopathy, and antioxidants may exert both preventive and therapeutic action. To evaluate the beneficial and antioxidant properties of food supplementation with Lisosan G, a powder of bran and germ of grain (Triticum aestivum) obtained by fermentation with selected lactobacillus and natural yeast strains, we used an in vivo model of hyperglycemia-induced retinal damage, the fruit fly Drosophila melanogaster fed with high-sucrose diet. Lisosan G positively affected the visual system of hyperglycemic flies at structural/functional level, decreased apoptosis, and reactivated protective autophagy at the retina internal network. Also, in high sucrose-fed Drosophila, Lisosan G reduced the levels of brain ROS and retina peroxynitrite. The analysis of oxidative stress-related metabolites suggested 7,8-dihydrofolate, uric acid, dihydroorotate, γ-L-glutamyl-L-cysteine, allantoin, cysteinyl-glycine, and quinolate as key mediators of Lisosan G-induced inhibition of neuronal ROS, along with the upregulation of glutathione system. Of note, Lisosan G may impact oxidative stress and the ensuing retinal cell death, also independently from autophagy, although the autophagy-ROS cross-talk is critical. This study demonstrated that the continuous supplementation with the alimentary integrator Lisosan G exerts a robust and multifaceted antioxidant effect on retinal neurons, thus providing efficacious neuroprotection of hyperglycemic eye.

Retinal damage in a new model of hyperglycemia induced by high-sucrose diets.

Loss of retinal neurons may precede clinical signs of diabetic retinopathy (DR). We studied for the first time the effects of hyperglycemia on the visual system of the fruit fly Drosophila melanogaster to characterize a model for glucose-induced retinal neurodegeneration, thus complementing more traditional vertebrate systems. Adult flies were fed with increased high-sucrose regimens which did not modify the locomotion ability, muscle phenotype and mobility after 10 days. The increased availability of dietary sucrose induced hyperglycemia and phosphorylation of Akt in fat tissue, without significant effects on adult growth and viability, consistent with the early phase of insulin signaling and a low impact on the overall metabolic profile of flies at short term. Noteworthy, high-sucrose diets significantly decreased Drosophila responsiveness to the light as a consequence of vision defects. Hyperglycemia did not alter the gross anatomical architecture of the external eye phenotype although a progressive damage of photosensitive units was observed. Appreciable levels of cleaved caspase 3 and nitrotyrosine were detected in the internal retina network as well as punctate staining of Light-Chain 3 and p62, and accumulated autophagosomes, indicating apoptotic features, peroxynitrite formation and autophagy turnover defects. In summary, our results in Drosophila support the view that hyperglycemia induced by high-sucrose diets lead to eye defects, apoptosis/autophagy dysregulation, oxidative stress, and visual dysfunctions which are evolutionarily conserved, thus offering a meaningful opportunity of using a simple in vivo model to study the pathophysiology of neuroretinal alterations that develop in patients at the early stages of DR.

Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy.

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous DysE17 and DysEP3397, lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.

Genome-Wide DNA Methylation and Gene Expression Profiles in Cows Subjected to Different Stress Level as Assessed by Cortisol in Milk.

Dairy cattle health, wellbeing and productivity are deeply affected by stress. Its influence on metabolism and immune response is well known, but the underlying epigenetic mechanisms require further investigation. In this study, we compared DNA methylation and gene expression signatures between two dairy cattle populations falling in the high- and low-variant tails of the distribution of milk cortisol concentration (MC), a neuroendocrine marker of stress in dairy cows. Reduced Representation Bisulfite Sequencing was used to obtain a methylation map from blood samples of these animals. The high and low groups exhibited similar amounts of methylated CpGs, while we found differences among non-CpG sites. Significant methylation changes were detected in 248 genes. We also identified significant fold differences in the expression of 324 genes. KEGG and Gene Ontology (GO) analysis showed that genes of both groups act together in several pathways, such as nervous system activity, immune regulatory functions and glucocorticoid metabolism. These preliminary results suggest that, in livestock, cortisol secretion could act as a trigger for epigenetic regulation and that peripheral changes in methylation can provide an insight into central nervous system functions.

Telomere length measurement in tumor and non-tumor cells as a valuable prognostic for tumor progression.

Telomere shortening has been supposed to be implicated in both aging and various human diseases especially carcinogenesis process. This phenomenon can lead to a chromosomal instability, contributing to a cell immortalization and tumor induction. In our study, we analyzed the role of telomere shortening in cancer progression, in Tunisian patients with digestive cancer. We measured the absolute telomere length in tumoral vs healthy adjacent tissues of each patient by using a q-RT PCR method and we investigated the relationship between telomere length and various sociodemographic and clinical parameters such as age, sex, tumor stage. In this pathological situation, we observed that, starting from 60 years of age, the telomere length increases in healthy mucosa and that in both healthy and cancer tissues, patients under 60 years have shorter telomeres, suggesting the telomere lengthening becomes more active with age. Finally, a positive correlation between normal and cancer tissues in both non-metastatic and metastatic stages, indicates telomere length in cancer tissue depends essentially on tumor stages. Our data allow us to suggest that telomere length depends on sex and age in healthy tissue while shortening and lengthening fluctuates considerably according to the tumor stage.

The Natural Compound Climacostol as a Prodrug Strategy Based on pH Activation for Efficient Delivery of Cytotoxic Small Agents.

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds.

CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.

Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in Drosophila melanogaster.

Lamin family proteins are structural components of a filamentous framework, the nuclear lamina (NL), underlying the inner membrane of nuclear envelope. The NL not only plays a role in nucleus mechanical support and nuclear shaping, but is also involved in many cellular processes including DNA replication, gene expression and chromatin positioning. Spermatogenesis is a very complex differentiation process in which each stage is characterized by nuclear architecture dramatic changes, from the early mitotic stage to the sperm differentiation final stage. Nevertheless, very few data are present in the literature on the NL behavior during this process. Here we show the first and complete description of NL behavior during meiosis and spermatogenesis in Drosophila melanogaster. By confocal imaging, we characterized the NL modifications from mitotic stages, through meiotic divisions to sperm differentiation with an anti-laminDm0 antibody against the major component of the Drosophila NL. We observed that continuous changes in the NL structure occurred in parallel with chromatin reorganization throughout the whole process and that meiotic divisions occurred in a closed context. Finally, we analyzed NL in solofuso meiotic mutant, where chromatin segregation is severely affected, and found the strict correlation between the presence of chromatin and that of NL.

Structural and Dynamic Characterization of the C313Y Mutation in Myostatin Dimeric Protein, Responsible for the "Double Muscle" Phenotype in Piedmontese Cattle.

The knowledge of the molecular effects of the C313Y mutation, responsible for the "double muscle" phenotype in Piedmontese cattle, can help understanding the actual mechanism of phenotype determination and paves the route for a better modulation of the positive effects of this economic important phenotype in the beef industry, while minimizing the negative side effects, now inevitably intersected. The structure and dynamic behavior of the active dimeric form of Myostatin in cattle was analyzed by means of three state-of-the-art Molecular Dynamics simulations, 200-ns long, of wild-type and C313Y mutants. Our results highlight a role for the conserved Arg333 in establishing a network of short and long range interactions between the two monomers in the wild-type protein that is destroyed upon the C313Y mutation even in a single monomer. Furthermore, the native protein shows an asymmetry in residue fluctuation that is absent in the double monomer mutant. Time window analysis on further 200-ns of simulation demonstrates that this is a characteristic behavior of the protein, likely dependent on long range communications between monomers. The same behavior, in fact, has already been observed in other mutated dimers. Finally, the mutation does not produce alterations in the secondary structure elements that compose the characteristic TGF-ß cystine-knot motif.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

BACKGROUND: Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. RESULTS: We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells. CONCLUSIONS: In vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

Microarray gene expression profiling of neural tissues in bovine spastic paresis.

BACKGROUND: Bovine Spastic Paresis (BSP) is a neuromuscular disorder which affects both male and female cattle. BSP is characterized by spastic contraction and overextension of the gastrocnemious muscle of one or both limbs and is associated with a scarce increase in body weight. This disease seems to be caused by an autosomal and recessive gene, with incomplete penetration, although no genes clearly involved with its onset have been so far identified. We employed cDNA microarrays to identify metabolic pathways affected by BSP in Romagnola cattle breed. Investigation of those pathways at the genome level can help to understand this disease. RESULTS: Microarray analysis of control and affected individuals resulted in 268 differentially expressed genes. These genes were subjected to KEGG pathway functional clustering analysis, revealing that they are predominantly involved in Cell Communication, Signalling Molecules and Interaction and Signal Transduction, Diseases and Nervous System classes. Significantly enriched KEGG pathway's classes for the differentially expressed genes were calculated; interestingly, all those significantly under-expressed in the affected samples are included in Neurodegenerative Diseases. To identify genome locations possibly harbouring gene(s) involved in the disease, the chromosome distribution of the differentially expressed genes was also investigated. CONCLUSIONS: The cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. By using this tool, we examined nearly 15000 transcripts and analysed gene pathways affected by the disease. Particularly, our data suggest also a defective glycinergic synaptic transmission in the development of the disease and an alteration of calcium signalling proteins. We provide data to acquire knowledge of a genetic disease for which literature still presents poor results and that could be further and specifically analysed in the next future. Moreover this study, performed in livestock, may also harbour molecular information useful for understanding human diseases.

Drosophila rae1 is required for male meiosis and spermatogenesis.

Previous studies of RAE1, a conserved WD40 protein, in Schizosaccharomyces pombe and mouse revealed a role in mRNA export and cell cycle progression in mitotic cells. Studies of RAE1 in Drosophila showed that the protein localizes to the nuclear envelope and is required for progression through the G1 phase of the cell cycle but not RNA export in tissue culture cells. Drosophila RAE1 also plays an essential developmental role, as it is required for viability and synaptic growth regulation as a component of an E3 ubiquitin ligase complex. Here we describe characterization of a new Drosophila rae1 mutant that is viable but results in male sterility. The mutant showed striking defects in primary spermatocyte nuclear integrity, meiotic chromosome condensation, segregation, and spindle morphology. These defects led to a failure to complete meiosis but allowed several aspects of spermatid differentiation to proceed, including axoneme formation and elongation. A GFP-RAE1 fusion protein that rescued most of the cytological defects showed a dynamic localization to the nuclear envelope, chromatin and other structures depending on the stage of spermatogenesis. A role for RAE1 in male meiosis, as well as mitotic cells, was also indicated by the defects induced by expression of rae1-RNAi. These studies in Drosophila provide the first evidence for an essential meiotic role of RAE1, and thus define RAE1 as a protein required for both meiotic and mitotic cell cycles.

Identification of a short region on chromosome 6 affecting direct calving ease in Piedmontese cattle breed.

Calving in cattle is affected by calf morphology and by dam characteristics. It is described by two different traits: maternal calving ease, which is the ability to generate dams with good physiological predisposition to calving, and direct calving ease, which is the ability to generate calves that are easily born. The aim of this study was to identify regions of cattle genome harboring genes possibly affecting direct calving ease in the Piedmontese cattle breed. A population of 323 bulls scored for direct calving ease (EBV) was analyzed by a medium-density SNP marker panel (54,001 SNPs) to perform a genome-wide scan. The strongest signal was detected on chromosome 6 between 37.8 and 38.7 Mb where 13 SNPs associated to direct calving ease were found. Three genes are located in this region: LAP3, encoding for a leucine aminopeptidase involved in the oxytocin hydrolysis; NCAPG, encoding for a non-SMC condensin I complex, which has been associated in cattle with fetal growth and carcass size; and LCORL, which has been associated to height in humans and cattle. To further confirm the results of the genome-wide scan we genotyped additional SNPs within these genes and analyzed their association with direct calving ease. The results of this additional analysis fully confirmed the findings of the GWAS and particularly indicated LAP3 as the most probable gene involved. Linkage Disequilibrium (LD) analysis showed high correlation between SNPs located within LAP3 and LCORL indicating a possible selection signature due either to increased fitness or breeders' selection for the trait.

Mealybug chromosome cycle as a paradigm of epigenetics.

Recently, epigenetics has had an ever-growing impact on research not only for its intrinsic interest but also because it has been implied in biological phenomena, such as tumor emergence and progression. The first epigenetic phenomenon to be described in the early 1960s was chromosome imprinting in some insect species (sciaridae and coccoideae). Here, we discuss recent experimental results to dissect the phenomenon of imprinted facultative heterochromatinization in Lecanoid coccids (mealybugs). In these insect species, the entire paternally derived haploid chromosome set becomes heterochromatic during embryogenesis in males. We describe the role of known epigenetic marks, such as DNA methylation and histone modifications, in this phenomenon. We then discuss the models proposed to explain the noncanonical chromosome cycle of these species.

Epigenetic marks for chromosome imprinting during spermatogenesis in coccids.

The establishment of sex-specific epigenetic marks during gametogenesis is one of the key feature of genomic imprinting. By immunocytological analysis, we thoroughly characterized the chromatin remodeling events that take place during gametogenesis in the mealybug Planococcus citri, in which an entire haploid set of (imprinted) chromosomes undergoes facultative heterochromatinization in male embryos. Building on our previous work, we have investigated the interplay of several epigenetic marks in the regulation of this genome-wide phenomenon. We characterized the germline patterns of histone modifications, Me(3)K9H3, Me(2)K9H3, and Me(3)K20H4, and of heterochromatic proteins, PCHET2 (HP1-like) and HP2-like during male and female gametogenesis. We found that at all stages in oogenesis chromatin is devoid of any detectable epigenetic marks. On the other hand, spermatogenesis is accompanied by a complex pattern of redistribution of epigenetic marks from euchromatin to heterochromatin, and vice versa. At the end of spermatogenesis, sperm heads are decorated by all the molecules we tested, except for PCHET2. However, only Me(3)K9H3 and Me(2)K9H3 are still detectable in the male pronucleus. We suggest that the histone H3 lysine 9 methylation is the signal used to establish the male-specific imprinting on the paternal genome, thus allowing it to be distinguished from the maternal genome in the developing embryo.

Microarrays and high-throughput transcriptomic analysis in species with incomplete availability of genomic sequences.

Microarrays produce a measurement of gene expression based on the relative measures of dye intensities that correspond to the amount of target RNA. This technology is fast developing and its application is expanding from Homo sapiens to a wide number of species, where enough information on sequences and annotations exist. Anyway, the number of species for which a dedicated platform exists is not high. The use of heterologous array hybridization, screening for gene expression in one species using an array developed for another one, is still quite frequent, even though cross-species microarray hybridization has raised many arguments. Some methods which are high throughput and do not rely on knowledge of the DNA/RNA sequence exist, namely serial analysis of gene expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and deep sequencing of full transcriptome. Although very powerful, particularly the latter, they are still quite costly and cumbersome methods. In some species where genome sequences are largely unknown, several anonymous sequences are deposited in gene banks as a result of Expressed Sequence Tags (ESTs) sequencing projects. The ESTs databases represent a valuable knowledge that can be exploited with some bioinformatic effort to build species-specific microarrays. We present here a method of high-density in situ synthesized microarrays starting from available EST sequences in, Ovis aries. Our data indicate that the method is very efficient and can be easily extended to other species of which genetic sequences are present in public databases, but neglected so far with advanced devices like microarrays. As a perspective, the approach can be applied also to species of which no sequences are available to date, thanks to high-throughput deep sequencing methods.