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Gian Carlo MANICARDI
CULTORE DELLA MATERIA
Dipartimento di Scienze della Vita
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Pubblicazioni
2022
- Composition and biological activity of essential oil from Artemisia roxburghiana Besser and Elsholtzia fruticosa Rehder cultivated in Italy
[Articolo su rivista]
Fusani, Pietro; Ronga, Domenico; Carminati, Domenico; Mandrioli, Mauro; Manicardi, Gian Carlo; Giannì, Sergio; Tava, Aldo
abstract
Artemisia roxburghiana Besser and Elsholtzia fruticosa Rehder plants were evaluated as innovative crops for the production of essential oil with valuable biological activity. A high essential oil content resulted for both species, that is 0.31% for A. roxburghiana and 0.46% for E. fruticosa. The composition of essential oil of A. roxburghiana is characterized by higher amount of cis-thujone, 1-8 cineole and camphor, while E. fruticosa oil is rich in 1,8-cineole and -terpinene. The biocidal activities of both oils were evaluated against some pathogenic Gram-positive and Gram-negative bacterial strains and as natural insecticides against aphids. E. fruticosa oil showed higher microbiologic activity compared to A. roxburghiana one, particularly against Staphylocosus aureus, Enterococcus faecium, Salmonella sp. and Escherichia coli. Both plant species revealed a high aphicidal activity against the polyphagous pest Myzus persicae, assessed by nymph mortality and fecundity reduction, and their efficacy resulted to be comparable to that of azadyrachtin. Due to the high content and the biocidal activity of these oils, the two investigated species represent a possible innovative crop for the production of essential oils to be employed in the food and the agro-industry.
2022
- The foraging gene is involved the presence of alate parthenogenetic females and explorative behaviours in the peach potato aphid Myzus persicae (Homoptera, Aphididae)
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
The foraging gene (for) encodes for a cyclic guanosine monophosphate (cGMP)-dependent protein kinase involved in behavioural plasticity in aphids and in other insects. In the present paper we analysed the complete for sequence in eight clones of the peach potato aphid Myzus persicae re-porting the presence of nonsense and frameshift mutations in the three studied clones character-ized by a reduced number of winged females and by the absence of exploratory behaviours. Quantitative PCR experiments evidenced similar results in clones possessing for genes with a conserved coding sequence, but low expression levels. The comparison of the for transcriptional level in Myzus persicae persicae and Myzus persicae nicotianae showed very different expression in the two studied M. p. nicotianae clones so that our data didn’t support a previous hypothesis suggesting that a differential for expression was related to ecological specialization of M. p. nico-tianae. In view of its role in both the dispersal of winged females and exploratory behaviours, the screening of the for sequences could be useful for predicting invasions of cultivated areas by peach potato aphids.
2021
- Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae
[Articolo su rivista]
Panini, M.; Chiesa, O.; Troczka, B. J.; Mallott, M.; Manicardi, G. C.; Cassanelli, S.; Cominelli, F.; Hayward, A.; Mazzoni, E.; Bass, C.
abstract
The evolution of resistance to insecticides threatens the sustainable control of many of the world's most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.
2020
- Evolutionary insights into the aphid genome: aphid genomics between quality problems and intriguing perspectives
[Capitolo/Saggio]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
In the last decade the genomes of several aphid species have been sequenced allow- ing a better understanding of their biology and evolution. Unfortunately, as frequently occurs with the next generation sequencing technologies, several aphid genomes con- sist in fragmented assemblies that contain thousands of genomic scaffolds of reduced length. In order to improve the quality of the published genomic data, several research groups are currently resequencing aphid DNA making possible to take the full advan- tage of genomics to face complex biological problems, such as aphid diversification. This review is aimed to discuss the current state of art in aphid genomics focusing in particular on the aspects that could improve our knowledge of their evolution.
2020
- Holocentric chromosomes
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Holocentric chromosomes possess multiple kinetochores along their length rather than the single centromere typical of other chromosomes. They have been described for the first time in cytogenetic experiments dating from 1935 and, since this first observation, the term holocentric chromosome has referred to chromosomes that: i. lack the primary constriction corresponding to centromere observed in monocentric chromosomes; ii. possess multiple kinetochores dispersed along the chromosomal axis so that microtubules bind to chromosomes along their entire length and move broadside to the pole from the metaphase plate. These chromosomes are also termed holokinetic, because, during cell division, chromatids move apart in parallel and do not form the classical V-shaped figures typical of monocentric chromosomes. Holocentric chromosomes evolved several times during both animal and plant evolution and are currently reported in about eight hundred diverse species, including plants, insects, arachnids and nematodes. As a consequence of their diffuse kinetochores, holocentric chromosomes may stabilize chromosomal fragments favouring karyotype rearrangements. However, holocentric chromosome may also present limitations to crossing over causing a restriction of the number of chiasma in bivalents and may cause a restructuring of meiotic divisions resulting in an inverted meiosis.
2020
- Soybean aphid biotype 1 genome: insights into the invasive biology and adaptive evolution of a major agricultural pest
[Articolo su rivista]
Giordano, Rosanna; Kiran Donthu, Ravi; Zimin, Aleksey; Consuelo Julca Chavez, Irene; Gabaldon, Toni; Munster, Manuellavan; Hon, Lawrence; Hall, Richard; Badger, Jonathan; Flores, Alejandra; Potter, Bruce; Giray, Tugru; Soto-Adames, Felipe N.; Weber, Everett; Marcelino, Jose A. P.; Fields, Christopher J.; J Voegtlin, David; Hill, Curt B.; Hartman, Glen L.; Akraiko, Tatsiana; Aschwanden, Andrew; Avalos, Arian; Band, Mark; Bonning, Bryony; Breault, Julie; Brier, Hugh; Chiesa, Olga; Chirumamilla, Anitha; Coates, Brad S.; Cocuzza, Giuseppe; Cullen, Eileen; Desborough, Peter; Diers, Brian; Di Fonzo, Christina; Gagnier, Dana; Gavloski, John; Marygebhardt, ; Hammond, Ronald B.; Heimpel, George; Herbert, Ames; Herman, Theresa; Hogg, David; Huanga, Yongping; Johnson, Doug; Knodel, Janet; Ko, Chiun-Cheng; Krupke, Christian H.; Labrie, Genevieve; Lagos-Kutz, Doris; Lang, Brian; Lee, Joon-Ho; Lee, Seunghwan; Mandrioli, Mauro; Manicardi, Gian Carlo; Maw, Eric L.; Mazzoni, Emanuele; Mccarville, Michael; Melchiori, Giulia; Michel, Andy; Micijevic, Ana; Miller, Nick; Mittenthal, Robin; Murai, Tamotsu; Nasruddin, Andy; Nault, Brian A.; O'Neal, Matthew E.; Panini, Michela; Pessino, Massimo; Prischmann-Voldseth, Deirdre; Quesnel, G.; Ragsdale, David W.; Robertson, Hugh H.; Schuster, Tiana; Sijun, Liu; Song, Hojun; Stimmel, James F.; Takahashi, Shigeru; Tilmon, Kelley; Tooker, John; Wilson, Sarah; Wu, Kongming; Zhan, Shuai; Yingzhang,
abstract
The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae) is a serious pest of the soybean plant, Glycine max, a major world-wide agricultural crop. We assembled a de novo genome sequence of Ap. glycines Biotype 1, from a culture established shortly after this species invaded North America. 20.4% of the Ap. glycines proteome is duplicated. These in-paralogs are enriched with Gene Ontology (GO) categories mostly related to apoptosis, a possible adaptation to plant chemistry and other environmental stressors. Approximately one-third of these genes show parallel duplication in other aphids. But Ap. gossypii, its closest related species, has the lowest number of these duplicated genes. An Illumina GoldenGate assay of 2,380 SNPs was used to determine the world-wide population structure of Ap. Glycines. China and South Korean aphids are the closest to those in North America. China is the likely origin of other Asian aphid populations. The most distantly related aphids to those in North America are from Australia. The diversity of Ap. glycines in North America has decreased over time since its arrival. The genetic diversity of Ap. glycines North American population sampled shortly after its first detection in 2001 up to 2012 does not appear to correlate with geography. However, aphids collected on soybean Rag experimental varieties in Minnesota (MN), Iowa (IA), and Wisconsin (WI), closer to high density Rhamnus cathartica stands, appear to have higher capacity to colonize resistant soybean plants than aphids sampled in Ohio (OH), North Dakota (ND), and South Dakota (SD). Samples from the former states have SNP alleles with high FST values and frequencies, that overlap with genes involved in iron metabolism, a crucial metabolic pathway that may be affected by the Rag-associated soybean plant response. The Ap. glycines Biotype 1 genome will provide needed information for future analyses of mechanisms of aphid virulence and pesticide resistance as well as facilitate comparative analyses between aphids with differing natural history and host plant range.
2019
- Analysis of the extent of synteny and conservation in the gene order in aphids: a first glimpse from the Aphis glycines genome
[Articolo su rivista]
Mandrioli, Mauro; Melchiori, Giulia; Panini, Michela; Chiesa, Olga; Giordano, Rosanna; Mazzoni, Emanuele; Manicardi, Gian Carlo
abstract
In the last decade several insect genomes have been sequenced, but for most the chromosomal mapping of the identified scaffolds/annotated genes is not available. The lack of this information makes it difficult to analyse various genetic aspects, including the presence of genome rearrangements and the extent of synteny within and across species. We mapped five multigenic DNA families (major and minor rDNAs, histone gene cluster, esterases and carotenoid desaturases) and seven scaffolds corresponding to 9 Mb of the soybean aphid, Aphis glycines, genome and identified loci spanning the four soybean aphid chromosomes. A comparative analysis of the localization of the annotated A. glycines genes with respect to the peach potato aphid, Myzus persicae, and the fly, Drosophila melanogaster, evidenced a lower degree of synteny between the two aphid species than in the aphid-fly comparison. Only 1.4 genes per syntenic block were observed in aphids in contrast to 2.3 genes per block in flies. This higher chromosomal rearrangement rate in aphids could be explained considering that they possess holocentric chromosomes that can favour the stabilization and inheritance of chromosomal rearrangements. Lastly, our experiments did not detect the presence of chimeric assemblies in the newly available A. glycines biotype 1 genome, differently from what reported in assembled genome of other aphid species, suggesting that chromosomal mapping can be used to ascertain the quality of assembled genomes.
2019
- Comparative analysis of intra- and inter-specific genomic variability in the peach potato aphid Myzus persicae
[Articolo su rivista]
Mandrioli, Mauro; Deborah, Salvatore; Ferrari, Agnese; Patelli, Niccolò; Manicardi, Gian Carlo
abstract
The availability of genomic data in the last decade relating to different aphid species has allowed the analysis of the genomic variability occurring among such species, whereas intra-specific variability has hitherto very largely been neglected. In order to analyse the intra-genomic variability in the peach potato aphid, Myzus persicae, comparative analyses were performed revealing several clone-specific gene duplications, together with numerous deletions/rearrangements. Our comparative approach also allowed us to evaluate the synteny existing between the two M. persicae clones tested and between the peach potato aphid and the pea aphid, Acyrthosiphon pisum. Even if part of the observed rearrangements are related to a low quality of some assembled contigs and/or to the high number of contigs present in these aphid genomes, our evidence reveals that aphid clones are genetically more different than expected. These results suggest that the choice of performing genomes sequencing combining different biotypes/populations, as revealed in the case of the soybean aphid, Aphis glycines, is unlikely to be very informative in aphids. Interestingly, it is possible that the holocentric nature of aphid chromosomes favours genome rearrangements that can be successively inherited transgenerationally via the aphid’s apomictic (parthenogenetic) mode of reproduction. Lastly, we evaluated the structure of the cluster of genes coding for the five histones (H1, H2A, H2B, H3 and H4) in order to better understand the quality of the two M. persicae genomes and thereby to improve our knowledge of this functionally important gene family
2019
- Potential role of the heat shock protein 90 (hsp90) in buffering mutations to favour cyclical parthenogenesis in the peach potato aphid Myzus persicae (Aphididae, Hemiptera)
[Articolo su rivista]
Mandrioli, Mauro; Elisa, Zanetti; Nardelli, Andrea; Manicardi, Gian Carlo
abstract
Heat-shock proteins 90 (hsp90s) are a class of molecules able to stabilize a network of “client” proteins that are involved in several processes. Furthermore, recent studies indicated that mutations in the hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden/cryptic mutations. In order to verify the role of hsp90 in aphids, we amplified and sequenced the hsp90 gene in seventeen lineages of the peach potato aphid Myzus persicae (Sulzer, 1776) looking for the presence of mutations. In particular, we compared lineages with different reproductive modes (obligate versus cyclical parthenogenesis), propensity to develop winged females and karyotype stability. Differently from the cyclical parthenogenetic lineages that possessed functional hsp90 genes, the seven analysed asexual lineages showed severe mutations (including frameshift and nonsense mutations). In vivo functional assays with the hsp90-inhibitor geldanamycin showed that some lineages with cyclical parthenogenesis may lose their ability to induce sexuales in the absence of active hsp90 revealing the presence of cryptic mutations in their genomes. As a whole, our data suggest that hsp90 could play in aphids a role in buffering hidden/cryptic mutations that disrupt cyclical parthenogenesis.
2019
- The evolutionary history and functional divergence of trehalase (treh) genes in insects
[Articolo su rivista]
Nardelli, Andrea; Vecchi, Matteo; Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Trehalases (treh) have been found in different organisms, such as bacteria, fungi, yeast, nematodes, insects, vertebrates and plants. Their biochemical properties are extremely variable and not yet fully understood. Gene expression patterns have shown differences among insect species suggesting a potential functional diversification of trehalase enzymes during their evolution. A second gene family encoding for enzymes with hypothetical trehalase activity has bene been repeatedly annotated in insect genome as acid trehalases/acid trehalase-like (ath), but its functional role is still not clear. The currently available large amount of genomic data from many insect species may enable deeper studies allowing a better understanding of the evolutionary history, the phylogenetic relationships and possible roles of trehalase encoding genes in this taxon. The aim of the present study is to infer the evolutionary history of trehalases and acid trehalase genes in insects and analyse the trehalase functional divergence during their evolution, combining nucleotides sequences phylogenyphylogenetic, protein conservation and genomic synteny/colinearity analyses.
2017
- Afit: a bioinformatic tool for measuring aphid fitness and invasiveness
[Articolo su rivista]
Nardelli, Andrea; Peona, V; Toschi, A; Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
A careful measure of fitness represents a crucial target in crop pest management and becomes fundamental considering extremely prolific insects. In the present paper, we describe a standardized rearing protocol and a bioinformatics tool to calculate aphid fitness indices and invasiveness starting from life table data. We tested the protocol and the bioinformatic tool using six Myzus persicae (Sulzer) asexual lineages in order to investigate if karyotype rearrangements and ecotype could influence their reproductive performances. The tool showed that different karyotypes do not influence adaptive success and put in evidence a marked invasive potential of the M. persicae lineage 64. The presence of a similar fitness rate of 33H and 7GK asexual lineages (both possessing intra-individual karyotype variations) in respect to the asexual lineage 1 (with a standard karyotype) represents an important demonstration of the potentiality of holocentric chromosomes to reduce the effects of chromosome rearrangements.
2017
- Analysis of the extent of shared synteny and conserved gene order in aphids: a first glimpse from Aphis glycines (Hemiptera, Aphididae) karyotype
[Poster]
Mandrioli, Mauro; Melchiori, Giulia; Panini, Michela; Chiesa, Olga; Mazzoni, Emanuele; Giordano, Rosanna; Manicardi, Gian Carlo
abstract
Recently, several insect genomes have been published, but most of them lack any information regarding the mapping of identified scaffolds, making impossible to evaluate both the presence of chromosomal rearrangements and sinteny among different taxa. Data regarding the chromosomal localization of the annotated genes could be, for instance, extremely relevant for the understanding of the evolution of the sex chromosomes and the sex determining system, a topic of great interest for pest crop insects.
Here we mapped 30 Mb of the Aphis glycines genome (whose genome sequencing has been recently completed) and compared the localization of the annotated genes to the genetic maps of the fly Drosophila melanogaster identifying orthologous loci spanning all the four A. glycines chromosomes. A. glycines is the first aphid species to have its genome, at least partially, mapped. The identification of several chromosome-specific markers will improve our possibility to explain the different physiological properties of A. glycines biotypes that currently represent a serious source of damages for soybean cultures in USA.
Indeed, A. glycines, as usual in aphids, possesses holocentric chromosomes that lack the primary constriction and cannot be banded with the traditional cytogenetic techniques so that the identification of chromosomal markers is essential for the proper investigation of the effects of the chromosomal rearrangements on aphid phenotypes.
2017
- Comparative gene mapping as a tool to understand the evolution of pest crop insect chromosomes
[Poster]
Mandrioli, Mauro; Zambonini, Giada; Manicardi, Gian Carlo
abstract
The extent of conservation of synteny and gene order in aphids has been previously investigated only by comparing a small subset of linkage groups between the pea aphid Acyrthosiphon pisum and few other aphid species. Here we compared the localization of eight A. pisum scaffolds (covering 5,3 Mb and 83 genes) in respect to the fly Drosophila melanogaster Muller elements identifying orthologous loci spanning all the four A. pisum chromosomes (as evidenced by FISH experiments).
Comparison of the genetic maps revealed that the gene order is not conserved, but a macroscale synteny is present across different loci suggesting that the study of the fly Muller elements could favour the identification of chromosomal markers useful for the study of chromosomal rearrangements in aphids.
The presence of chromosomal portions with homology between aphids and Diptera is particularly relevant in view of the holocentric nature of the aphid chromosomes, suggesting that, although holocentrism could favour local rearrangement, the overall gene content of the chromosomes is not altered.
A. pisum is the first aphid species to have its genome sequenced and it has been suggested a substantial synteny of gene order and orientation with other Aphidinae so that the finding of several chromosomal regions in synteny between Diptera and Hemiptera indicates that the genomic tools developed in A. pisum will be broadly useful also for the study of other aphid species of relevant agricultural interest.
2017
- Comparative gene mapping as a tool to understand the evolution of pest crop insect chromosomes
[Articolo su rivista]
Mandrioli, Mauro; Zambonini, Giada; Manicardi, Gian Carlo
abstract
The extent of the conservation of synteny and gene order in aphids has been previously
investigated only by comparing a small subset of linkage groups between the pea aphid
Acyrthosiphon pisum and a few other aphid species. Here we compared the localization of eight
A. pisum scaffolds (covering more than 5 Mb and 83 genes) in respect to the Drosophila melanogaster
Muller elements identifying orthologous loci spanning all the four A. pisum chromosomes.
Comparison of the genetic maps revealed a conserved synteny across different loci suggesting
that the study of the fruit fly Muller elements could favour the identification of chromosomal markers
useful for the study of chromosomal rearrangements in aphids. A. pisum is the first aphid species to
have its genome sequenced and the finding that there are several chromosomal regions in synteny
between Diptera and Hemiptera indicates that the genomic tools developed in A. pisum will be
broadly useful not only for the study of other aphids but also for other insect species.
2016
- An overview of the main pathways of metabolic resistance in insects
[Articolo su rivista]
Panini, M; Manicardi, Gian Carlo; Moores, Gd; Mazzoni, E.
abstract
Insecticides have played and still fulfil a very important role in sustainable production of food, animal feed and also as protection against disease vectors. They act to suppress insect populations and, as a consequence of their use, insecticide resistance has evolved. An overview of insecticide resistance mechanisms in insects is given, focusing on the metabolic systems involved in xenobiotic metabolism in the class Insecta. Several enzyme families (e.g., esterases, mixed function oxidases, glutathione S-transferases) are involved in insecticide detoxification, sequestration and excretion and have differing relative importance within the various taxonomic groups. A brief discussion of their impact on control strategies is given.
2016
- Ecological genetics of Italian peach-potato aphid (Myzus persicae)
populations in relation to geography, dispersal and insecticide
resistance as studied using microsatellite and resistance markers
[Articolo su rivista]
Monti, Valentina; Loxdale, Hugh D.; Cesari, Michele; Frattini, Milo; Panini, Michela; Mazzoni, Emanuele; Manicardi, Gian Carlo
abstract
1 Eight polymorphic microsatellites and two insecticide resistance markers (kdr and
R81T) were used to investigate the population genetics and demography of resistance
mechanisms in field populations of the peach-potato aphid, Myzus persicae (Sulzer),
a global crop pest, in several areas of Italy, notably the north-east, aiming to
determine whether these parameters are linked to population structuring resulting from
insecticide selective sweeps.
2 Individuals collected directly from peach orchards (main primary host) in the
spring, especially after control failure, revealed 90 microsatellite alleles, of which
approximately one-third were rare.
3 Many populations deviated from Hardy–Weinberg expectations, in a few instances
as a result of null alleles. FIS values were on average negative (indicating heterozygote
excess), whereas FST values (mean=0.137) reflected moderate interpopulation
gene flow, with nonstructured populations having an estimated higher level
than insecticide-treated ones. Population assignment and Mantel testing showed a
non-homogenous pattern, with some populations isolated genetically. Linkage disequilibrium
was detected in four of the microsatellites and both resistance markers.
Contrasting FST patterns with resistance status supported the view that some populations
were indeed structured.
4 Neighbour-joining trees revealed some populations to have similar insecticide resistance
profiles, independent of geographical origin.
5 Overall, the findings reveal that the natural M. persicae populations investigated in
the present study, living within an intensively cultivated agro-ecosystem, bear the
signature of such management in terms of population structuring, as well as possibly
also dynamics.
2016
- Genomic and cytogenetic localization of the carotenoid genes in the aphid genome
[Articolo su rivista]
Mandrioli, Mauro; Rivi, V; Nardelli, Andrea; Manicardi, Gian Carlo
abstract
Data published in the scientific literature suggests a possible link between chromosomal rearrangements involving autosomes 1 and 3 and the presence of red morphs in the peach-potato aphid Myzus persicae (Sulzer). In order to begin a study of this relationship, we analysed the genomic and chromosomal location of genes involved in carotenoid biosynthesis in M. persicae and the pea aphid, Acyrthosiphon pisum (Harris), since carotenoids are the basis of the colour in many aphid species. Genomic analysis identified a DNA sequence containing carotenoid genes in synteny between the 2 species. According to the results obtained using in situ PCR, carotenoid genes were located in a subterminal portion of autosome 1 in both species. The same localization has also been observed in the onion aphid Neotoxoptera formosana Takahashi that, as M. persicae and A. pisum, belongs to the tribe Macrosiphini, thereby suggesting a synteny of this chromosomal region in aphids. In situ PCR experiments performed on 2 M. persicae asexual lineages bearing heterozygous translocations involving autosomes 1 and 3 revealed that carotenoid genes were located within chromosomal portions involved in recurrent rearrangements. We also verified by bioinformatics analyses the presence of fragile sites that could explain these recurrent rearrangements in M. persicae.
2016
- Role of ant-tending in modulating the presence of symbiotic bacteria against parasitoids in aphids
[Articolo su rivista]
Mandrioli, Mauro; Bisanti, Matteo; Grasso, Da; Manicardi, Gian Carlo
abstract
Several species of parasitoid wasps have been used to control aphids in the field in order to improve the environmental sustainability of the current agricultural practices. Behavioural and physiological defences have been reported in aphids and the role of symbiotic bacteria, such as Hamiltonella defensa, Serratia symbiotica and Regiella insecticola, has been molecularly dissected to understand their protection against hymenopteran parasitoids. Interestingly, the relationship between the presence of defensive symbionts and the type of aphid-ant mutualism (obligate, facultative or absent) is still understudied, whereas ants could strongly influence the ecological costs of defensive symbionts. In the present paper, we performed a screening of 16 aphid species collected in Italy and compared the presence and abundance of defensive symbionts to the degree of myrmecophily of the sampled aphid populations revealing that Hamiltonella defensa is the most common defensive symbiont. Our data suggest a direct effect of ant-tending on the abundance of H. defensa such that if aphids are maintained in insectaries in the absence of ants, aphids increase the amount of this defensive symbiont making the composition of their microbiome context-dependent.
2015
- Cytosine methylation in insects: new routes for the comprehension of insect complexity
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Cytosine methylation is one of the most studied epigenetic modifications and its occurrence has been deeply studied in mammals and plants. DNA methylation (together with other epigenetic modifications of DNA and histones) plays an important role in different processes. Indeed, several morphological and/or behavioural traits may origin as a consequence of the epigenetic modulation of genes so that identical genes can results in different “morphs”. Despite considerable progress during recent years, many questions remain since it is largely unknown how the environment triggers alterations in the epigenome. In the present review we discuss the use of aphids and honey bees as epigenetic experimental model to understand how cytosine methylation is directly or indirectly linked to environmental factors. Indeed, the epigenetic changes of DNA could be at the basis of unexpected morphological differences explaining also complex traits.
2015
- Environmental hexachlorobenzene exposure and human malereproductive function
[Articolo su rivista]
Specht, Io; Bonde, Jp; Toft, G; Giwercman, A; Spanò, M; Bizzaro, D; Manicardi, Gian Carlo; Jönsson, Bag; Robbins, Wa
abstract
Hexachlorobenzene (HCB) is a persistent environmental fungicide that may disrupt
androgen regulation. The aim of this study was to investigate associations between
HCB levels and biomarkers of male reproductive function. 589 spouses of pregnant
women from Greenland, Poland and Ukraine were enrolled between 2002 and 2004.
The men provided semen and blood samples and were interviewed. HCB was
measured in serum by gas chromatography. The mean serum concentrations of HCB
were higher in Ukraine (182.3 ng/g lipid) and Greenland (79.0 ng/g lipid) compared to
Poland (14.2 ng/g lipid). Sex Hormone Binding Globulin (SHBG) and Free Androgen
Index (FAI) were associated with HCB in men from Ukraine and Poland.
This study spanning large differences in environmental HCB exposure levels shows a
positive association for SHBG and negative association for FAI with high serum levels
of HCB in fertile men, but without major consequences for semen quality and the Inuit
study population.
2015
- Fast chromosomal evolution and karyotype instability: recurrent chromosomal rearrangements in the peach potato aphid Myzus persicae (Hemiptera: Aphididae)
[Articolo su rivista]
Manicardi, Gian Carlo; Nardelli, Andrea; Mandrioli, Mauro
abstract
The occurrence of karyotype variations with respect to both chromosome number and structure has been frequently reported in aphids. Here, we review recent data attesting to the presence of recurrent chromosomal changes in the karyotype of the peach potato aphid Myzus persicae, where clones presenting metaphases with different chromosome number (from 12 to 17) have been observed, also comparing plates obtained within the same embryo. According to the available data, M. persicae autosomes 3 and 1 are the chromosomes mostly involved in changes compared to other autosomes, suggesting that they could have sites more susceptible to fragmentation. Chromosomal fissions involving the X chromosomes have also been observed, suggesting that they may have fragile sites located at the termini opposite to the nucleolar organizer regions-bearing telomere. The presence of holocentric chromosomes and reproduction by apomictic parthenogenesis, together with a constitutive expression of telomerase, could explain the inheritance of the observed chromosomal instability in aphids. Considering that chromosomal changes may affect the host choice and could also favour speciation, it would be intriguing to confirm whether the observed karyotype variants have effects over short temporal and spatial scales.
2015
- Presence of kdr and s-kdr resistance in Musca domestica populations collected in Piacenza province (Northern Italy)
[Articolo su rivista]
Mazzoni, Emanuele; Chesa, Olga; Puggioni, Vincenzo; Panini, Michela; Manicardi, Gian Carlo; Bizzaro, Davide
abstract
Pyrethroid insecticides combine a high efficacy against insects with a low toxicity towards warm-blooded vertebrates. For this
reason they are largely used to control housefly infestations. The efficacy of these products is affected by insecticide resistance
mechanisms. In particular, some mutations in the sodium channel coding sequence are responsible for target-site resistance to
pyrethroid insecticides. This work presents a new molecular approach based on allele-specific PCRs to point out and to
characterise this resistance mechanism in two M. domestica populations (PNT and TRV) collected near Piacenza (Northern Italy).
The presence of different kdr and s-kdr genotypes was assessed for population PNT whilst kdr only was detected in population
TRV. Dose-response bioassays evidenced quite high resistance factors, expecially for population PNT. This is in line with the kdr
and s-kdr frequencies observed in the assayed populations.
2015
- The cytogenetic architecture of the aphid genome
[Articolo su rivista]
Manicardi, Gian Carlo; Mandrioli, Mauro; Blackman, Rl
abstract
In recent years aphids, with their well-defined polyphenism, have become favoured as model organisms for the study
of epigenetic processes. The availability of the pea aphid (Acyrthosiphon pisum) genome sequence has engendered much
research aimed at elucidating the mechanisms by which the phenotypic plasticity of aphids is inherited and controlled.
Yet so far this research effort has paid little attention to the cytogenetic processes that play a vital part in the organisation,
expression and inheritance of the aphid genome. Aphids have holocentric chromosomes, which have very different
properties from the chromosomes with localised centromeres that are found in most other organisms. Here we review
the diverse forms of aphid chromosome behaviour that occur during sex determination and male and female meiosis,
often in response to environmental changes and mediated by endocrine factors. Remarkable differences occur, even
between related species, that could have significant effects on the inheritance of all or parts of the genome. In relation
to this, we review the particular features of the distribution of heterochromatin, rDNA genes and other repetitive DNA
in aphid chromosomes, and discuss the part that these may play in the epigenetic modification of chromatin structure
and function.
2014
- Aphids (hemiptera)
[Capitolo/Saggio]
Mandrioli, M.; Manicardi, G. C.
abstract
Aphids (Hemiptera: Aphididae) are ancient insects, whose fossils go back to the Triassic, about 220-210 million years (Grimaldi and Engel 2005). To date, they are present in most of the world’s biomes, including the tropics and subarctic regions, where they infest a huge range of plants (Loxdale 2009) causing several damages due not only to their direct parasitic action against crops, but also because they are active vectors of numerous crop viruses (Blackman and Eastop 2000, 2006, 2007; van Emden and Harrington 2007).
2014
- Exposure to polybrominated diphenyl ethers and male reproductive function in Greenland, Poland and Ukraine
[Articolo su rivista]
Gunnar, Toft; Virissa, Lenters; Roel, Vermeulen; Dick, Heederik; Cathrine, Thomsen; Georg, Becher; Aleksander, Giwercman; Davide, Bizzaro; Manicardi, Gian Carlo; Marcello, Spanò; Lars, Rylander; Henning S., Pedersen; Paweł, Strucinski; Valentyna, Zviezdai; Jens Peter, Bonde
abstract
tAnimal and a few human studies suggest that polybrominated diphenyl ethers (PBDEs) may affect malereproductive function. The aim of the present study was to evaluate if male reproductive function wasassociated with serum level of PBDEs. We evaluated, in a cross-sectional study, the effects of environmen-tal exposure to BDE-47 and BDE-153 on reproductive hormones and semen quality, including markersof DNA damage and apoptosis, in 299 spouses of pregnant women from Greenland, Poland and Ukraine.Adjusted linear regression models indicated no strong associations between BDE-47 or BDE-153 expo-sure and markers of male semen quality or reproductive hormones. In the largest study to date wedemonstrate that BDE-47 and BDE-153 exposure was not associated with altered semen characteristicsor reproductive hormones, indicating that male reproductive function is not affected by the exposurelevel of these compounds in fertile European or Arctic populations.
2014
- Interactions between polymorphisms in the aryl hydrocarbon receptor signalling pathway and exposure to persistent organochlorine pollutants affect human semen quality.
[Articolo su rivista]
Brokken LJS, 1. 0. 2.; Lundberg, Pj; Spanò, M; Manicardi, Gian Carlo; Pedersen, Hs; Struciński, P; Góralczyk, K; Zviezdai, V; Jönsson, Bag; Bonde, Jp; Toft, G; Lundberg Giwercman, Y; Giwercman, A.
abstract
Persistent
organic
pollutants
(POPs)
may
affect
male
reproductive
function.
Many
dioxin-like
POPs
exert
their
effects
by
activation
of
the
aryl
hydrocarbon
receptor
(AHR)
signalling
pathway.
We
analysed
whether
gene–environment
interactions
between
polymorphisms
in
AHR
(R554K)
and
AHR
repressor
(AHRR
P185A)
and
serum
levels
of
markers
of
POP
exposure
1,1-bis-(4-chlorophenyl)-2,2-dichloroethene
(
p,p
-DDE)
and
2,2
,4,4
,5,5
-hexachlorobiphenyl
(CB-153)
are
associated
with
21
parameters
of
male
reproductive
function
in
581
proven-fertile
European
and
Greenlandic
men.
In
Greenlandic
men,
AHR
variants
significantly
modified
the
association
between
serum
levels
of
both
p,p
-DDE
and
CB-153
and
inhibin
B
levels,
sperm
chromatin
integrity,
and
seminal
zinc
levels.
In
the
total
cohort,
interactions
between
AHRR
variants
and
serum
levels
of
CB-153
were
associated
with
sperm
chromatin
integrity
and
the
expression
of
the
pro-apoptotic
marker
protein
Fas.
The
data
indicate
that
susceptibility
to
adverse
effects
of
POP
exposure
on
male
reproductive
function
is
dependent
on
polymorphisms
in
genes
involved
in
AHR
signalling
2014
- Karyotype rearrangements and telomere analysis in Myzus persicae (Hemiptera, Aphididae) strains collected on Lavandula sp. plants
[Articolo su rivista]
Mandrioli, Mauro; F., Zanasi; Manicardi, Gian Carlo
abstract
Karyotype analysis of nine strains of the peach-potato aphid Myzus persicae (Sulzer, 1776), collected on Lavandula sp. plants, evidenced showed that five of them had a standard 2n = 12 karyotype, one possessed a fragmentation of the X chromosome occurring at the telomere opposite to the NOR-bearing one and three strains had a chromosome number 2n = 11 due to a non-reciprocal translocation of an autosome A3 onto an A1 chromosome. Interestingly, the terminal portion of the autosome A1 involved in the translocation was the same in all the three strains, as evidenced by FISH with the histone cluster as a probe. The study of telomeres in the M. persicae strain with the X fission evidenced that telomerase synthesised de novo telomeres at the breakpoints resulting in the stabilization of the chromosomal fragments. Lastly, despite the presence of a conserved telomerase, aphid genome is devoid of genes coding for shelterin, a complex of proteins involved in telomere functioning frequently reported as conserved in eukaryotes. The absence of this complex, also confirmed in the genome of other arthropods, suggests that the shift in the sequence of the telomeric repeats has been accompanied by other changes in the telomere components in arthropods in respect to other metazoans.
2014
- Mapping the aphid genome: the cytogenetic dimension of a pest crop insect.
[Capitolo/Saggio]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
n.d.
2014
- Occurrence of a Rabl-like telomere clustering in the holocentric chromosome of the peach potato aphid Myzus persicae (Hemiptera; Aphididae).
[Articolo su rivista]
Mandrioli, Mauro; Bandinelli, S; Manicardi, Gian Carlo
abstract
Several studies demonstrated that chromosome anchoring to nuclear structures is involved in the organization of the interphase nucleus. The Rabl configuration, a well-studied chromosome organization in the interphase nucleus, has been deeply studied in organisms with monocentric chromosomes but just slightly touched in species with holocentric chromosomes. In the present paper, by means of the isolation and chromosomal mapping of the C0t DNA fraction and chromatin immunoprecipitation with anti-LEM-2 antibodies, we evidenced the presence of few foci where telomeres and subtelomeric regions cluster in the aphid interphase nuclei, suggesting the occurrence of a Rabl-like chromosome configuration. The same experimental approaches also evidenced that most of the repetitive DNA of the 2 X chromosomes is located at the periphery of the nucleus, whereas the ribosomal genes, located at 1 telomere of each X chromosome, are present towards the inner portion of the nucleus, favoring their transcriptional activity.
2014
- Recent changes in the distribution of carboxylesterase genes and associated chromosomal rearrangements in Greek populations of the tobacco aphid Myzus persicae nicotianae. Biological Journal of the Linnean Society, 113: 455-470.
[Articolo su rivista]
Kati, A. N.; Mandrioli, Mauro; P. J., Skouras; G. L., Malloch; C., Voudouris; M., Venturelli; Manicardi, Gian Carlo; J. A., Tsitsipis; B., Fenton; J. T., Margaritopoulos
abstract
We present data on the frequency of amplified E4 and FE4 carboxylesterase genes in Myzus persicae s.l. clones collected during the years 2002–2007 and 2012 in Greece. Most clones were of the tobacco aphid, Myzus persicae nicotianae. Samples from 2012 were genotyped with microsatellite DNA markers and a number of them were karyotyped. Aphid clones with amplified FE4 genes predominated in all years, whereas E4 was present in only 3.5% of all samples and always occurred in clones with FE4. Most of the clones examined showed high carboxylesterase activity levels (R2 resistant category). The results showed marked changes in the frequencies of the two carboxylesterase genes in the tobacco aphid populations compared to published data that were collected in Greece in the mid 1990s, when E4 was recorded on its own in 20% of all samples and in 32% of samples from tobacco. A parallel change in karyotype was also observed because the A1,3 translocation, which had a worldwide association with amplified E4 genes in the 1990s, was not detected in the clones analyzed in 2012. Possible causes for these changes are discussed, although selection as a result of pest management practices appears to be the major one. Novel chromosomal rearrangements were also found in M. persicae nicotianae clones. These rearrangements could be a result of clastogenic effects of nicotine, which could persist because of the holocentric nature of aphid chromosomes. The results are discussed in relation to rapid evolution events that have taken place in the tobacco aphid in Greece during the last two decades
2013
- A novel in vitro sperm head decondensation protocol for a rapid flow cytometric measurement of DNA content
[Articolo su rivista]
Antonucci N, 9. 5.; Manes, S; Corradetti, B; Manicardi, Gian Carlo; Borini, A; Bizzaro, D.
abstract
Objective: To set up a novel protocol of sperm head in vitro decondensation that obviates the problematic effect of the variable degree
of sperm chromatin packaging on DNA staining needed for flow cytometric analysis.
Design: Development of a new cytofluorimetric assay.
Setting: University laboratory.
Patient(s): Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences,
Universita Politecnica delle Marche.
Intervention(s): Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry
to measure DNA content.
Main Outcome Measure(s): Mean fluorescent channel, DNA content, percentage diploid sperm.
Result(s): Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated Cvalue
(approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by
fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier.
Conclusion(s): This study establishes a simple method for in vitro sperm head decondensation,
which allows accurate detection of the real sperm DNA content
2013
- A1-3 chromosomal translocations in Italian populations of the peach potato aphid Myzus persicae (Sulzer) not linked to esterase-based insecticide resistance
[Articolo su rivista]
Rivi, Marco; Monti, Valentina; E., Mazzoni; Cassanelli, Stefano; M., Panini; M., Anaclerio; M., Ciglini; B., Corradetti; D., Bizzaro; Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Esterase-based resistance in the peach-potato aphid, Myzus persicae (Sulzer), is generally due to one of two alternative amplified carboxylesterase genes, E4 or FE4 (fast E4). The E4 amplified form is distributed worldwide and it is correlated with a
particular translocation between autosomes 1 and 3, whereas the FE4 form, which has hitherto not been found to be associated with chromosomal rearrangements, is typical of the Mediterranean regions. In this study, we present for the first time cytogenetic and molecular data on some M. persicae parthenogenetic lineages, which clearly show a chromosomal A1-3 translocation associated with esterase FE4 genes and unrelated to high levels of esterase-based resistance.
2013
- Bis (2-ethylexhyl) phthalate impairs spermiogenesis in zebrafish (Danio rerio)
[Articolo su rivista]
Corradetti, B; Stronati, A; Tosti, L; Manicardi, Gian Carlo; Carnevali, O; Bizzaro, D.
abstract
Bis-(2-ethylhexyl) phthalate (DEHP) is a widely used industrial additive for increasing plastic
!exibility. Its metabolites are known to exert toxic effects on reproduction and development
of mammals. The aim of this study was to evaluate the effects of environmentally relevant
concentrations of DEHP (0.2 and 20 mg/L) on the reproductive biology of adult male zebra"sh
(Danio rerio). The effects of DEHP and 17b-ethynylestradiol (a positive control) were determined
after one or three weeks of exposure by TUNEL assay, histomorphometric analysis
and evaluation of reproductive performance. DEHP impaired reproduction in zebra"sh by
inducing a mitotic arrest during spermatogenesis, increasing DNA fragmentation in sperm
cells and markedly reducing embryo production (up to 90%). In conclusion, relatively shortterm
exposure to environmentally relevant concentrations of DEHP is able to alter spermatogenesis
and affect reproduction in zebra"sh.
2013
- Characterization of Non-LTR Retrotransposable TRAS Elements in the Aphids Acyrthosiphon pisum and Myzus persicae (Aphididae, Hemiptera)
[Articolo su rivista]
V., Monti; C., Serafini; Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
A non-LTR TRAS retrotransposon (identified as TRASAp1) has been amplified in the pea aphid Acyrthosiphon pisum and its presence has been assessed also in the peach potato aphid Myzus persicae. This TRAS element possesses 2 overlapping ORFs (a gag-ORF1 and a pol-ORF2 containing the reverse transcriptase and the endonuclease domains) that show a similarity ranging from 40% to 48% to proteins coded by other TRAS elements identified in insects (including the beetle Tribolium castaneum and the moth Bombyx mori). The study of the TRAS chromosomal insertion sites, performed by standard fluorescent in situ hybridization (FISH) and fiber FISH, showed that TRAS elements were located in a subtelomeric position, just before the telomeric (TTAGG)n repeats. In both the aphid species, TRAS elements were present at all termini of autosomes, but the 2 X chromosome telomeres show a clear-cut structural difference. Indeed, cromomycin A3 staining, together with FISH using a TRAS probe, revealed that TRAS signals only occur at the telomere opposite to the NOR-bearing one. Lastly, the analysis of the distribution of TRAS retrotransposons in a M. persicae strain possessing spontaneous fragmentations of the X chromosomes assessed that TRAS elements were not involved in the healing of de novo telomeres.
2013
- Chromosomal mapping reveals a dynamic organization of the histone
genes in aphids (Hemiptera: Aphididae)
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Despite their involvement in different processes, histone genes have been analysed in few insects. In order to improve the knowledge about this important gene family, genes coding for histones have been analysed in the aphid Acyrthosiphon pisum showing that at the amino acid level, aphid histones are highly conserved. In particular, data from A. pisum confirm that H1 is the most variable of the five histones,
whereas histones H3 and H4 are highly conserved with the H3 almost identical from insects to vertebrates. A. pisumhistone genes are organized in a quintet with the H1 gene followed by H2A and H2B genes that are adjacent and transcribed in same directions, in the opposite strand in respect to the H1 gene. At the 3’ end of the histone cluster, genes H3 and H4 constitute an oppositely transcribed pair. The span of the aphid histone genes (more than 7 kb) is greater than the average length of
the histone cluster till now reported in insects (about 5 kb).
Furthermore, spacers that separate the aphid histone genes vary in length. The histone genes have been mapped in A. pisum and successively in the aphids Myzus persicae and Rhopalosiphum padi showing that they are present in a single large cluster located in an interstitial position of autosomes 1, differently from what reported in the Russian wheat aphid Diuraphis noxia,where histone genes have been localized in a telomere of the two X chromosomes suggesting a dynamic organization of this multigene family in aphids.
2013
- Evolving aphids: one genome-one organism insects or holobionts?
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
Aphids have obligate mutualistic relationships with microorganisms that provide them with
essential substances lacking in their diet, together with symbionts conferring them conditional adaptive
advantages related, for instance, to the thermal tolerance and to the resistance to parasitoid wasps.
The presence/absence of a secondary symbiont may have a relevant phenotypic effect so that aphid
microbial symbionts constitute a sort of second genome with its own genetic inheritance. On the
whole, genes important for aphid survival and reproduction are not uniquely present in the aphid
nuclear and mitochondrial genomes, but also in the chromosomes of each symbiont. As a
consequence, aphids should be viewed as holobionts with an extended genome (the hologenome)
including the host and its symbiotic microbiome. In this connection, the true unit of selection in
evolution must be considered the aphid holobiont, in place of the single host as individual separated
from its symbionts
2013
- LA RESISTENZA AGLI INSETTICIDI NEI LEPIDOTTERI: CYDIA POMONELLA COME CASO STUDIO
[Relazione in Atti di Convegno]
Manicardi, Gian Carlo
abstract
The codling moth, Cydia pomonella (Cp) is a major pest in most pome fruit orchards worldwide. Under the selective pressure
caused by an increasing number of chemical treatments, Cp has developed a reduced sensitivity towards different pesticides.
In this paper, we overviewed the mechanisms of insecticide resistance in Cp populations with particular emphasis on
data collected in Emilia-Romagna orchards showing that the reduced susceptibility to insecticide treatments seems to be mainly
caused by an increased MFO activity with a minor contribution of GST. Target-site mutations within AChE-1 seems a rare
phenomenon world wide, since they were found in Spain only. Given the complexity of the mechanisms of resistance found so
far in Cp, only the availability of breeding population that can be maintained under selection pressure, will allow to explore
the genetic basis of resistance with greater chance of success.
The understanding of the molecular basis of the resistance mechanisms is the starting point in order to allow the adoption of
novel pest management strategies directed to circumvent and/or delay resistance outbreaks and to control the insensitive pests.
2013
- Non-linear association between androgen receptor CAG and GGN repeat lengths and reproductive parameters in fertile European and Inuit men.
[Articolo su rivista]
9. 6. Brokken L. J., S.; Rylander, L; Jönsson, B; Spanò, M; Pedersen, Hs; Ludwicki, Jk; Zviezdai, V; Bizzaro, D; Manicardi, Gian Carlo; Toft, G; Bonde, Jp; Giwercman, A; Lundberg Giwercman, Y.
abstract
Recently the dogma that there is an inverse linear association between androgen receptor (AR) CAG and GGN polymorphisms and receptor activity has been challenged. We analysed the pattern of association between 21 male reproductive phenotypes and AR CAG/GGN repeat lengths in 557 proven-fertile men.A linear association was only found between sperm DNA fragmentation index (DFI) and CAG length, and between inhibin B and GGN length. Men with longer CAG then the reference (22-24), had higher oestradiol levels, whereas men with shorter CAG stretches had a higher DFI and a higher proportion of Fas-positive germ cells. Subjects with either short or long CAG had increased seminal levels of prostate-specific antigen and neutral α-glucosidase activity. Compared to men with the median GGN length of 23, those with shorter GGN repeats had higher levels of inhibin B, higher proportions of normal and progressive sperm, and a higher fraction of Fas-positive sperm, while men with longer GGN had higher oestradiol levels.These data indicate that at least for some markers of male reproductive function the association with CAG or GGN repeat length is curvilinear.
2012
- Biochemical, molecular and field monitoring
of insecticide resistance in codling moth populations
collected in Emilia Romagna (Italy) orchards
[Relazione in Atti di Convegno]
M., Rivi; Cassanelli, Stefano; A., Butturini; E., Pasqualini; M., Boselli; S., Civolani; S., Caruso; Manicardi, Gian Carlo
abstract
Codling moth populations from Emilia-Romagna orchards with difficulties in pest control
have been studied combining biochemical and molecular tests, bioassays and small scale field trials.
Biochemical assays aimed to evaluate EST, GST and MFO activities, put in evidence that the field
populations examined have mainly developed a MFO detoxifying response, in some cases supported
by EST and/or GST contributes. A direct sequencing strategy of the AChE-1 gene showed that none of
the analyzed strains possessed target-site mutations within AChE-1 sequence. Field trials and
bioassays revealed that increased MFO was frequently associated to a reduction of IGR sensibility
(tebufenozide and diflubenzuron) and, in less extend, to chlorpyrifos.
2012
- Continuous occurrence of intra-individual chromosome rearrangements in the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)
[Articolo su rivista]
Monti, Valentina; G., Lombardo; H. D., Loxdale; Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
Analysis of the holocentric mitotic chromosomes of the peach-potato aphid, Myzus persicae (Sulzer), from clones labelled 50, 51 and 70 revealed different chromosome numbers, ranging from 12 to 14, even within each embryo, in contrast to the standard karyotype of this species (2n = 12). Chromosome length measurements, combined with fluorescent in situ hybridization experiments, showed that the observed chromosomal mosaicisms are due to recurrent fragmentations of chromosomes X, 1 and 3. Contrary to what has generally been reported in the literature, X chromosomes were frequently involved in recurrent fragmentations, in particular at their telomeric ends opposite to the nucleolar organizer region. Supernumerary B chromosomes have been also observed in clones 50 and 51. The three aphid clones showed recurrent fissions of the same chromosomes in the same regions, thereby suggesting that the M. persicae genome has fragile sites that are at the basis of the observed changes in chromosome number. Experiments to induce males also revealed that M. persicae clones 50, 51 and 70 are obligately parthenogenetic, arguing that the reproduction by apomictic parthenogenesis favoured the stabilization and inheritance of the observed chromosomal fragments.
2012
- Karyotype variations in Italian populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae).
[Articolo su rivista]
Rivi, Marco; Monti, Valentina; E., Mazzoni; Cassanelli, Stefano; M., Panini; D., Bizzaro; Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
In this study, we present cytogenetic data regarding 66 Myzus persicae strainscollected in different regions of Italy. Together with the most common 2n= 12karyotype, the results showed different chromosomal rearrangements: 2n= 12 withA1–3 reciprocal translocation, 2n= 13 with A1–3 reciprocal translocation and A3fission, 2n= 13 with A3 fission, 2n= 13 with A4 fission, 2n= 14 with X andA3 fissions. A 2n= 12–13 chromosomal mosaicism has also been observed.Chromosomal aberrations (and in particular all strains showing A1–3 reciprocaltranslocation) are especially frequent in strains collected on tobacco plants,and we suggest that a clastogenic effect of nicotine, further benefited by theholocentric nature of aphid chromosomes, could be at the basis of the observedphenomenon
2012
- Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics
[Articolo su rivista]
Ina O., Specht; Marcello, Spanò; Karin S., Hougaard; Manicardi, Gian Carlo; Davide, Bizzaro; Gunnar, Toft; Aleksander, Giwercman; Jens Peter E., Bonde
abstract
Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ cell population to numbers that can be supported by the Sertoli cells, and, possibly, selective depletion of meiotic and postmeiotic abnormal germ cells. We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men. Six hundred and four men from Greenland, Poland and Ukraine were consecutively enrolled during their pregnant wife's antenatal visits. Semen analysis was performed as recommended by the World Health Organization. Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell. DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The percentage of Fas-positive sperm cells was higher in men with high total sperm count (P<0.01), more motile sperms (P=0.04) and fewer sperm head defects (P=0.05). These associations were consistent within and across study regions. Furthermore, testosterone, follicle-stimulating hormone (FSH) and sexual hormone-binding globulin (SHBG) were significantly negatively correlated with Fas within and across regions as well. The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics. The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarify the biological significance of sperm apoptotic markers.
2012
- Sperm DNA integrity in relation to exposure to environmental perfluoroalkyl substances – A study of spouses of pregnant women in three geographical regions.
[Articolo su rivista]
Olmer Specht I, 9. 1.; Hougaard, Ks; Spanò, M; Bizzaro, D; Manicardi, Gian Carlo; Lindh, Cg; Toft, G; Jönsson, Bag; Giwercman, A; Bonde, Jp
abstract
Perfluoroalkyl substances (PFASs) can interfere with male reproductive function, but evidence in humansis limited. Six hundred four fertilemen(199 from Greenland, 197 from Poland and 208 from Ukraine) wereenrolled in the study. We measured four PFASs in serum (PFOS, PFOA, PFNA and PFHxS) and concurrentDNA damage in spermatozoa by sperm chromatin structure assay (SCSA) and in situ terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay, apoptotic markers in semen (Fas-receptorand Bcl-xL), and reproductive hormones in serum. No association between PFASs and SCSA, apoptoticmarkers or reproductive hormones emerged.Weobserved a slight increase in SHBG and TUNEL-positivitywith increased PFOA exposure in men from Greenland. Thus, consistent evidence that PFAS exposureinterferes with sperm DNA fragmentation, apoptosis or reproductive hormones was not found.
2012
- Starting at the end: telomeres and telomerase in arthropods
[Articolo su rivista]
Mandrioli, Mauro; Monti, Valentina; Manicardi, Gian Carlo
abstract
Telomere composition and structure have been studied in several arthropods allowing us to better understand the evolution of such an important portion of the eukaryotic chromosomes. Genes coding for telomerase reverse transcriptase (TERT) have been sequenced and studied in few arthropod species only,where they resulted highly transcribed also in somatic tissues suggesting a different TERT regulation in respect to vertebrates. Contrary to the strict conservation of telomeres,subtelomeric regions were more polymorphic and heterogeneous in composition and frequently contained retrotransposable elements that strongly influenced subtelomere evolution.
2012
- The vanishing clone: karyotypic evidence for extensive intraclonal genetic variation in the peach potato aphid, Myzus persicae (Hemiptera:Aphididae)
[Articolo su rivista]
Monti, Valentina; Mandrioli, Mauro; Rivi, Marco; Manicardi, Gian Carlo
abstract
Analysis of holocentric mitotic metaphase chromosomes of the peach-potato aphid Myzus persicae (Sulzer) clone 33H revealed different chromosome numbers, ranging from 12 to 17 within each embryo, in contrast to the standard karyotype of this species (2n=12). Chromosome length measurements revealed that the observed chromosomal mosaicism is due to recurrent fragmentations of chromosomes X, 1 and 3 due to fragile sites or to hot spots of recombination. Fluorescent in situ hybridization experiments showed that X chromosomes were frequently involved in recurrent fragmentations, in particular their telomeric end opposite to the nucleolar organizer region (NORs). Experiments to induce males showed that M. persicae clone 33H is obligately parthenogenetic. The reproduction by apomictic parthenogenesis, together with a high telomerase expression that stabilized the chromosomes involved in the fragmentations observed in the M. persicae clone 33H, appears to favour the stabilization of the observed chromosome instability.
2012
- Unlocking Holocentric Chromosomes: New Perspectives from Comparative and Functional Genomics?
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
The presence of chromosomes with diffuse centromeres (holocentric chromosomes) has been reported in several taxa since more than fifty years, but a full understanding of their origin is still lacking. Comparative and functional genomics are nowadays furnishing new data to better understand holocentric chromosome evolution thus opening new perspectives to analyse karyotype rearrangements in species with holocentric chromosomes in particular evidencing unusual common features, such as the uniform GC content and gene distribution along chromosomes.
2011
- Basic and clinical aspects of Sperm Chromomycin A3 assay.
[Capitolo/Saggio]
Manicardi, Gian Carlo; Bizzaro, D; Sakkas, D.
abstract
Semen quality is conventionally determined according to the number, motility, and morphology ofspermatozoa in an ejaculate. In turn, it is generally accepted that an association exists between these semenparameters and fertilizing ability. With the advent of in vitro fertilization (IVF) and related techniquessuch as intracytoplasmic sperm injection (ICSI), it has become increasingly apparent that the number,motility, and morphology of spermatozoa are not always indicative of a male’s fertility status. Methodsexploring sperm DNA stability and integrity have been applied during the last decade to evaluate fertilitydisorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. It hasbeen shown that infertile men have an increased sperm histone–protamine ratio than fertile counterparts.This alteration of histone–protamine ratio, also called abnormal packing, increases susceptibility of spermDNA to external stresses due to poorer chromatin compaction. Recent studies have also underlined the linkbetween protamine deficiency and sperm DNA damage that resulted in poor fertilizing capacity.
2011
- Composition and Epigenetic Markers of Heterochromatin in the Aphid Aphis nerii (Hemiptera: Aphididae).
[Articolo su rivista]
Mandrioli, Mauro; Azzoni, Paola; G., Lombardo; Manicardi, Gian Carlo
abstract
A detailed karyotype analysis of the oleander aphid Aphis nerii focusing on the distribution, molecular composition and epigenetic modifications of heterochromatin was done in order to better understand the structure and evolution of holocentric/holokinetic chromosomes in aphids. The female karyotype (2n = 8) consisted of 3 pairs of autosomes and a pair of X chromosomes that were the longest elements in the karyotype and carried a single, terminally located nucleolar organizer region. Males showed 2n = 7 chromosomes due to the presence of a single X chromosome. Heterochromatin was located in the X chromosomes only and consisted of 4 satellite DNAs that have been identified. A. nerii constitutive heterochromatin was enriched in mono-, di- and tri-methylated H3 histones and HP1 proteins but, interestingly, it lacked DNA methylation that was widespread in euchromatic chromosomal regions. These results suggest that aphid heterochromatin is assembled and condensed without any involvement of DNA methylation.
2011
- Cytogenetic and molecular analysis of the holocentric chromosomes of the potato aphid Macrosiphum euphorbiae (Thomas, 1878)
[Articolo su rivista]
Monti, Valentina; Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
Cytogenetic and molecular investigations on the holocentric chromosomes of the aphid Macrosiphum euphorbiae (Thomas, 1878) have been carried out using silver staining and C-banding (followed by chromomycin A3 and DAPI staining) in order to improve our knowledge about the structure of aphid chromosomes. C-banding pattern is peculiar since only the two X chromosomes and a single couple of autosomes presented heterochromatic bands. Silver staining and FISH with the 28S rDNA probe localized the rDNA genes on one telomere of each X chromosome that also resulted brightly fluorescent after chromomycin A3 staining of C-banded chromosomes, whereas all other heterochromatic bands were DAPI positive. Interestingly, a remarkable nucleolar organizing region (NOR) heteromorphism was present making the two X chromosomes easily distinguishable. Southern blotting and FISH assessed the presence of the (TTAGG)n repeat at the ends of all the M. euphorbiae chromosomes. Karyotype analysis showed that all males possessed the X chromosome with the larger amount of rDNA suggesting a non-Mendelian inheritance of the two X chromosomes.
2011
- Presence of a functional (TTAGG)n telomere-telomerase system in aphids
[Articolo su rivista]
Monti, Valentina; M., Giusti; D., Bizzaro; Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
The structure of the telomeres of four aphid species (Acyrthosiphon pisum, Megoura viciae, Myzus persicae and Rhopalosiphum padi) has been evaluated by Southern blotting and fluorescent in situ hybridization. This revealed that each chromosomal end consists of the (TTAGG)n repeat. The presence of a telomerase coding gene has been successively verified in the A. pisum genome, revealing that aphid telomerase presents a sequence identity ranging from 12 to 18% with the invertebrate and vertebrate homologues and possesses the two main domains involved in telomerase activity. Interestingly, telomerase expression has been verified in different somatic tissues suggesting that in aphids the telomerase activity could be not restricted as in human cells. The study of telomeres in a M. persicae strain with variable chromosome number evidenced that aphid telomerase can initiate the de novo synthesis of telomere sequences at internal breakpoints resulting in the stabilization of the chromosomal fragments.
2011
- The stability of instability: occurrence of a functional telomerase in the holocentric chromosomes of aphids
[Abstract in Atti di Convegno]
Monti, Valentina; Mandrioli, Mauro; M., Rivi; D., Bizzaro; Manicardi, Gian Carlo
abstract
nn
2010
- BIOCHEMICAL AND MOLECULAR MONITORING OF INSECTICIDE RESISTANCE IN CODLING MOTH POPULATIONS COLLECTED IN EMILA-ROMAGNA (ITALY) ORCHARDS.
[Abstract in Atti di Convegno]
Rivi, Marco; Cassanelli, Stefano; E., Pasqualini; S., Civolani; A., Butturini; M., Boselli; Manicardi, Gian Carlo
abstract
The codling moth, Cydia pomonella is a major pest in most pome fruit orchards worldwide. Recently, under the selective pressure caused by an increasing number of chemical treatments the codling moth has progressively developed a reduced insecticide susceptibility primarily to organophosphates (acetylcholinesterase inhibitors), and to insect growth regulators (chitin synthesis inhibitors). Small-scale field trials along with bioassays and biochemical or molecular tests are often exploit to reveal the selection of resistance mechanisms in field, such as target site insensibility and enhanced detoxifying activities. In this vein direct AChE-1 gene sequencing and biochemical analysis (EST, GST and MFO activities) were performed in codling moth populations collected in several Emilia Romagna orchards having pest control difficulties. In all analysed populations, the reduced susceptibility to insecticide treatments seems mainly associated to an increased MFO activity while GST and EST detoxifying pathways are more rarely involved. Moreover electrophoretic profiles, show that EST isoforms may be detected in some OP resistant population. These differences may also occur in GST and MFO families, for which there are no currently available rapid qualitative test. It must be stressed that none of the examined populations possess target-site mutations within AChE-1 sequence. The prevalent metabolic profile emerging from our analyses results in agreement with that found in other European codling moth populations with a reduced susceptibility to pesticide treatments based on alternate use of organophosphates and IGR. Trials were carried out to evaluate the correlations between the laboratory results and the field responses of two codling moth populations.
2010
- Distribution and molecular composition of heterochromatin in the holocentric chromosomes of the aphid Rhopalosiphum padi (Hemiptera: Aphididae)
[Articolo su rivista]
Monti, Valentina; Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
In order to study the structure of holocentric chromosomes in aphids, the localization and the composition of Rhopalosiphum padi heterochromatin and rDNA genes have been evaluated at cytogenetic and molecular level. In particular, heterochromatin resulted located on all the chromosomes both in intercalary and telomeric positions. Moreover, enzymatic digestion of R. padi genome put in evidence a DraI satellite DNA which has been isolated, cloned and sequenced. FISH experiments showed that this satellite DNA clusters in an intercalary C-positive band on the two X chromosomes.
2010
- Mechanisms of environmental reproductive toxicity: Sperm DNA damage
[Relazione in Atti di Convegno]
D., Bizzaro; Manicardi, Gian Carlo
abstract
The spermatogenetic process in the human produces a heterogeneous cell population showing different degrees of maturation, variable morphological features and fertilizing capacity. There is a large body of evidence that some of the ejaculated spermatozoa possess a variety of abnormalities at the nuclear, cytoskeletal, and organelle levels and that these anomalies impact fertility. Damage of sperm DNA or its chromatin structure can occur at any step of whole spermatogenesis and could have endogenous (i. e. pathological altered germ cell maturation) or exogenous origin (i. e. exposure to xenobiotic substances). Three main theories have been proposed to explain DNA anomalies in the ejaculated human spermatozoa (Sakkas and Alavarez, 2010). The first theory supports that DNA damage in mature spermatozoa is associated with poor chromatin packaging or abnormal packing due to underprotamination and/or alteration of Topisomerase II activity which results in the presence of endogenous nicks in DNA (Manicardi et al., 1995). The second theory proposes that sperm DNA damage could be related to a process called “abortive apoptosis” whereby sperm cells are earmarked for apoptosis during spermatogenesis but survive to be present in the ejaculate (Sakkas et al., 2002). A major factor affecting sperm is also their vulnerability to oxidative stress, because they are deficient in both antioxidant and DNA-repair systems and rich in targets for oxidative attack (Aitken et al., 1998). The correct DNA packaging and stability appears to be fundamental for the protection against both endogenous and exogenous DNA damage. Transmission of damaged DNA to the offspring, particularly at levels that exceed the DNA repair capacity of the oocyte, has been shown to have serious consequences in animal models and is also being more and more implicated in the human. Normal sexual development, differentiation and function in the human has been shown to be altered by the presence of synthetic chemicals in the environment acting as endocrine disruptors or mutation agents. Moreover, exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, either individually or together, can affect the integrity of sperm chromatin (Stronati et al., 2006). The observation that ejaculated human spermatozoa possess DNA damage raises numerous problems relating to why and how these spermatozoa arise in the ejaculate of some men and what consequences they have if they succeed in their genetic project. Understanding the mechanisms responsible will improve our knowledge about certain causes of male infertility.
2010
- Set up of a novel in-vitro sperm head decondensation protocol for a rapid flow cytometric measurement of the dna content
[Abstract in Rivista]
Antonucci, N; Stronati, A; Manes, S; Corradetti, B; Manicardi, Gian Carlo; Borini, A; Bizzaro, D.
abstract
The paper illustrate a method able to allow a novel in-vitro sperm head decondensation protocol for a rapid flow cytometric measurement of the DNA content which could be usefull in male infertility investigations.
2010
- Survey of Susceptibility to Abamectin of Pear Psylla (Hemiptera: Psyllidae) in Northern Italy.
[Articolo su rivista]
S., Civolani; Cassanelli, Stefano; Rivi, Marco; Manicardi, Gian Carlo; R., Peretto; M., Chicca; E., Pasqualini; M., Leis
abstract
The pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), is a relevant pest of pear, Pyrus communis L., trees in Emilia-Romagna region (northern Italy). The susceptibility to the insecticide abamectin was evaluated at different times of the year on C. pyri populations undergoing different control strategies within conventional, integrated, and organic farms. The tests performed were the egg spray and the topic and dip bioassay on adults. The larval mortality was evaluated by dip bioassay on treated leaves. The activity of P450-dependent monooxygenases, a relevant enzyme system involved in insecticide resistance of C. pyri, was also determined in adults by 7-ethoxycoumarin O-deethylation (ECOD assay). Tests on treated eggs and on larvae showed no significant differences in LC50 and LC90, although these values were always lower in individuals collected from organic farms in comparison with all other farms. Tests on overwintering adults revealed differences among populations, probably more related to collection time than to field pest control strategies. Unexpectedly, the ECOD assay on adults showed a slightly higher cytochrome P450 monooxygenase activity in the population undergoing organic control in comparison to others. Our results indicate that egg spray is the most reliable bioassay to verify data of open-field applications. Apparently, no resistance to abamectin has yet been developed by C. pyri in Emilia-Romagna.
2010
- Un nuovo protocollo di decondensazione del DNA negli spermatozoi in vitro, per una corretta e rapida valutazione del DNA content in citofluorimetria a flusso.
[Abstract in Atti di Convegno]
N., Antonucci; A., Stronati; S., Manes; B., Corradetti; Manicardi, Gian Carlo; A., Borini; D., Bizzaro
abstract
il contributo descrive un nuovo protocollo di decondensazione del DNA negli spermatozoi in grado di permettere una corretta e rapida valutazione del DNA content in citofluorimetria a flusso.
2009
- Distribution of heterochromatin and rDNA on the holocentric chromosomes of the aphids Dysaphis plantaginea and Melanaphis pyraria (Hemiptera: Aphididae)
[Articolo su rivista]
Angela, Criniti; Gabriele, Simonazzi; Cassanelli, Stefano; Mario, Ferrari; Davide, Bizzaro; Manicardi, Gian Carlo
abstract
The structure of the holocentric chromosomes of the rosy apple aphid, Dysaphis plantaginea (2n = 12), and pear-grassaphid, Melanaphis pyraria (2n = 8), was studied using C-banding, NOR, Giemsa and fluorochrome staining, and fluorescent in situhybridization (FISH). Contrary to the equilocal distribution of heterochromatin typical of monocentric chromosomes, in both species C-banding evidenced a tendency of highly repetitive DNAs to be restricted to the X chromosomes. Silver staining and FISH, using a 28S rDNA probe, located rDNA genes on one telomere of each X chromosome, the only brightly fluorescent C-positive sites revealed by CMA3 staining, whereas all other heterochromatic C-bands were DAPI positive. Both species showed a noticeable amount of rDNA heteromorphism. Mitotic recombination is proposed as a possible mechanism responsible for the variation in size of rDNA.
2009
- Heterochromatin and rdna localization on the holocentric chromosomes of black bean aphid, aphis fabae scop. (hemiptera: Aphididae)
[Articolo su rivista]
Rivi, M.; Cassanelli, Stefano; Mazzoni, E.; Bizzaro, D.; Manicardi, Gian Carlo
abstract
Heterochromation and rDNA localization in the holocentric chromosomes of the aphid Aphis fabae has been investigated at a cytological level after C-banding, Giemsa, DAPI, CMA3 and NOR staining. C-banding technique showed that heterochromatic areas result mainly clustered on the X chromosomes but, contrary to what observed in other aphid species, in A. fabae C-positive bands are clearly distinguishable also on each autosomal chromosome pair, thus allowing the identification of homologues and a reliable reconstruction of the karyotype. Moreover, silver staining localized rDNA genes on one telomere of each X chromosome; these are the only brightly fluorescent C-positive regions revealed after CMA3 staining, whereas all other heterochromatic bands are DAPI positive. Both CMA3 and silver staining evidenced a noticeable amount of rDNA heteromorphism.
2009
- INSECTICIDE RESISTANCE IN ITALIAN POPULATIONS OF THE PEACH POTATO APHID MYZUS PERSICAE (SULZER) (HEMIPTERA, APHIDIDAE).
[Articolo su rivista]
Cravedi, P.; Manicardi, Gian Carlo; Cassanelli, Stefano; Talesa, V.; Delbuono, C.; Bizzaro, D.; Mazzoni, E.
abstract
Research on insecticide resistance in Myzus persicae (Sulzer) in Italy was started by the Institute of Entomologyand Plant Pathology of the “Università Cattolica del Sacro Cuore” in Piacenza (Northern Italy) in the early 1990s. Nowcontinuously reared cultures of about 100 populations, collected mainly from Italian peach orchards, are available.Insecticide resistance mechanisms in these populations have been characterised. Close collaboration with other Italianresearchers was started to combine different research techniques. This has allowed the discovery in the sodium channelgene of a new mutation (F979S) that increases resistance to pyrethroid insecticides. Esterase- and modified acetylcholinesterase-based resistance has also been investigated.
2009
- RELATIONSHIP BETWEEN CHROMOSOMAL TRANSLOCATION AND FE4 GENE AMPLIFICATION IN AN ITALIAN POPULATION OF THE PEACH POTATO APHID MYZUS PERSICAE (HEMIPTERA: APHIDIDAE).
[Articolo su rivista]
Rivi, M.; E., Mazzoni; A., Criniti; Cassanelli, Stefano; D., Bizzaro; Manicardi, Gian Carlo
abstract
In this study we present cytogenetic data attesting the identification of an Italian Myzus persicae (Sulzer) strain possessing an A 1, 3 chromosomal translocation and we analyse the relationships between this chromosomal re-arrangement and insecticide resistance by means of biochemical and DNA-based diagnostic assays.
2008
- Analisi biochimiche e molecolari applicate allo studio della resistenza agli agrofarmaci di carpocapsa in Emila-Romagna
[Relazione in Atti di Convegno]
Cassanelli, Stefano; V., Farinelli; Anceschi, Elena; A., Butturini; L., Caroli; Manicardi, Gian Carlo
abstract
La carpocapsa (Cydia pomonella) è senza dubbio il fitofago più dannoso per le pomacee. La lotta integrata a questo lepidottero può realizzarsi attraverso l’uso concertato del virus, della confusione sessuale e di trattamenti insetticidi, prevalentemente a base di organofosforici (OPs) e inibitori della crescita (IGRs). Tuttavia a partire dall’ultimo decennio, si è riscontrata una crescente difficoltà nel contenere gli attacchi di carpocapsa, e sebbene il numero dei trattamenti sia aumentato, non sempre la difesa ha avuto successo. In questo lavoro sono illustrati i risultati relativi al monitoraggio della resistenza agli insetticidi OPs e IGRs in popolazioni emiliano-romagnole di C. pomonella, mediante l’impiego combinato di analisi biochimiche, volte a saggiare le principali attività detossificanti, e di analisi genetiche, che prevedono la ricerca di eventuali mutazioni nel gene che codifica per l’acetilcolinestarasi 1 (AChE-1, resistenza target site). I dati sinora ottenuti, dimostrano una certa diffusione di resistenza basata sui sistemi detossificanti e, tra essi, un prevalente coinvolgimento dei sistemi enzimatici glutatione-S-transferasi (GST) e monossigenasi (MFO), mentre il contributo delle esterasi (EST) sembra più marginale. Al contrario, nelle popolazioni di C. pomonella analizzate, non sono emerse mutazioni target site a carico del gene AChE-1.
2008
- Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)
[Articolo su rivista]
Criniti, A; Mazzoni, E; Cassanelli, Stefano; Cravedi, P; Tondelli, A; Bizzaro, D; Manicardi, Gian Carlo
abstract
In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central-Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its paratype sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.
2008
- DNA break induction and meiotic impairment in Zebrafish spermatogenesis after bis(2-ethylhexyl)phthalate (DEHP) exposure.
[Abstract in Atti di Convegno]
B., Corradetti; A., Stronati; L., Tosti; N., Antonucci; S., Manes; E., Proietti; M., Murano; Manicardi, Gian Carlo; C., Rubini; O., Carnevali; D., Bizzaro
abstract
In this abstract we present data attesting an influence of bis(2-ethylhexyl)phthalate (DEHP) exposure on DNA break induction and meiotic impairment in Zebrafish spermatogenesis.
2008
- Fertility and markers of male reproductive function in Inuit and European populations spanning large contrasts in blood levels of persistent organochlorines.
[Articolo su rivista]
Bonde, Jp; Toft, G; Rylander, L; RIGNELL HYDBOM, A; Giwercman, A; Spano, M; Manicardi, Gian Carlo; Bizzaro, D; Ludwicki, Jk; Zvyezday, V; BONEFELD JORGENSEN, Ec; Pedersen, Hs; Jonsson, Bag; Thulstrup, A. M.
abstract
BJECTIVE: We synthesized the main findings from an international epidemiologic study on the impact of biopersistent organic pollutants (POPs) on human reproductive function. DATA SOURCES AND EXTRACTION: We used a database with interview and biological data from 2,269 women and their spouses, and 18 published core papers. DATA SYNTHESIS: The study did not provide direct evidence of hormone-like activity of the polychlorinated biphenyl (PCB) congener CB-153 and the main dichlorodiphenyltrichloroethane (DDT) metabolite, 1 1 1 -dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p'-DDE), as serum concentrations of these compounds were not consistently related to either endogenous or exogenous hormone activity in serum. Nevertheless several links bewteen POP exposure and biomarkers of male reproductive function were identified. First, an association between high CB-153 serum levels and low sperm counts was detected within a subgroup of men with short androgen receptor CAG repeat length. Second, a relationship between increased CB-153 serum concentrations and decreased sperm motility was seen in all four studied regions, and indications of reduced neutral alpha-glucosidase activity in seminal plasma point to a post-testicular effect. Third, damage of sperm chromatin integrity was considerably less frequent in Greenlandic Inuits compared with that in European groups, and only in the latter was impairment of sperm chromatin integrity related to POPs. Despite these effects, fertility in terms of time taken to conceive was not related to POPs except in Inuits. A likely explanation of the latter was not identified. CONCLUSIONS: POPs may interfere with male reproductive function without major impact on fertility. The data do not provide direct evidence for endocrine disruption, hence other mechanisms should also be considered.
2008
- Human sperm DNA flow cytometry: preliminar results of new procedures for rapid genotoxic assay as Sperm-Flow-FISH
[Abstract in Atti di Convegno]
N., Antonucci; E., Proietti; A., Stronati; S., Manes; B., Corradetti; M., Murano; Manicardi, Gian Carlo; D., Bizzaro
abstract
This contribution deals with a presentation of a procedure of human sperm DNA flow cytometry aimed to allow a rapid genotoxic assay as Sperm-Flow-FISH
2008
- Human sperm genomic integrity: which information from different assays?
[Abstract in Atti di Convegno]
S., Manes; N., Antonucci; A., Stronati; E., Proietti; B., Corradetti; M., Murano; M., Cignitti; Manicardi, Gian Carlo; D., Bizzaro
abstract
This paper compares different methods utilized for the evaluation of human sperm genomic integrity and illustrated their possible application in the field of artificilal reproduction techniques (ART)
2008
- POP’s exposition in Swedish fishermen: effect on sperm quality
[Abstract in Atti di Convegno]
A., Stronati; G., Medi; E., Proietti; S., Manes; N., Antonucci; B., Corradetti; M., Murano; B. A. G., Jönsson; A., Rignell Hydbom; L., Rylander; A., Giwercman; G., Toft; J. P., Bonde; Bonefeld Jorgensen, E. C.; Manicardi, Gian Carlo; D., Bizzaro
abstract
The paper deals with an evaluation of the relationships between POP’s exposition and sperm quality (evaluated by means of Tunel assay and apoptotic markers) in Swedish fishermen:
2008
- Relation between serum xenobiotic induced receptor activities and DNA damage and sperm apoptotic markers in European and Inuit populations
[Abstract in Rivista]
Long, M.; Stronati, A.; Bizzaro, D.; Kruger, T.; Manicardi, Gian Carlo; Hjelmborg, P. S.; Spano, M.; Giwercman, A.; Toft, G.; Bonde, J. P.; Bonefeld Jorgensen, E. C.
abstract
Persistent organic pollutants (POPs) can interfere with hormone activities and are suspected as endocrine disrupters involved in, e.g. reproductive disorders. We investigated the possible relation between the actual integrated serum xenoestrogenic, xenoandrogenic and aryl hydrocarbon receptor activities and the sperm DNA damage and sperm apoptotic markers of 262 adult males (54 Inuits from Greenland, 69 from Warsaw (Poland), 81 from Sweden and 58 from Kharkiv (Ukraine) exposed to different levels of POPs.
2008
- Survey on the presence of “MACE” and “kdr” mutations in populations of the green peach aphid (Myzus persicae) from Emilia-Romagna peach orchards: preliminary results
[Relazione in Atti di Convegno]
Pavesi, F; Criniti, A; Cassanelli, Stefano; Bizzaro, D; Manicardi, Gian Carlo; Cravedi, P; Mazzoni, E.
abstract
The green peach aphid (Myzus persicae), is lastly one of well studied insect model todissect insecticide resistance mechanisms. As a result, a few in-vitro diagnostic tools were added to thetraditional in-vivo bioassays to monitor the presence of resistance in populations of this pest. Sincedifferent control strategies and different resistance mechanisms are often involved, both biochemicaland molecular approaches must be followed. Biochemical assays can be used to asses esterasedetoxifying/sequestering activity as well as the acetylcholinesterase insensitivity to some insecticides.Molecular screening by RFLP-PCR, may be also effectively performed to detect previously identifiedtarget site mutations. Herewith we analyzed aphid populations collected in peach orchards fromEmilia-Romagna (the most important peach growing area in Italy) for the presence of elevated esteraseexpression and acetylcholinesterase insensitivity to pirmicarb. Gene mutation occurrence inacetylcholinesterase (S431F) and in voltage-dependent sodium channel (kdr), conferring resistance todimethylcarbammates.and pyretroids respectively, have been also.checked. Information regarding theco-selection of different insecticide resistance mechanisms are thought supporting technical assistanceservices, to select the most suitable pest management strategies against M. persicae.
2008
- Xenobiotic activity in serum and sperm chromatin integrity in European and Inuit populations
[Articolo su rivista]
Kruger, T; Spano, M; Long, M; Eleuteri, P; Rescia, M; Hjelmborg, Ps; Manicardi, Gian Carlo; Bizzaro, D; Giwercman, A; Toft, G; Bonde, Jp; BONEFELD JORGENSEN, E. C.
abstract
Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AIR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans.
2007
- Androgen receptor gene CAG repeat length as a modifier of the association between persistent organohalogen pollutant exposure markers and semen characteristics.
[Articolo su rivista]
Giwercman, A; Rylander, L; RIGNELL HYDBOM, A; Jonsson, Ba; Pedersen, Hs; Ludwicki, Jk; Lesovoy, V; Zvyezday, V; Spano, M; Manicardi, Gian Carlo; Bizzaro, D; BONEFELD JORGENSEN, Ec; Toft, G; Bonde, Jp; Giwercman, C; Tiido, T; Giwercman, Yl
abstract
Objectives Exposure to persistent organohalogen pollutants was suggested to impair male reproductive function. A gene-environment interaction has been proposed. No genes modifying the effect of persistent organohalogen pollutants on reproductive organs have yet been identified. We aimed to investigate whether the CAG and GGN polymorphisms in the androgen receptor gene modify the effect of persistent organohalogen pollutant exposure on human sperm characteristics. Methods Semen and blood from 680 men [mean (SD) age 34 (10) years] from Greenland, Sweden, Warsaw (Poland) and Kharkiv (Ukraine) were collected. Persistent organohalogen pollutant exposure was assessed by measuring serum levels of 2,2,4,4,5,5'-hexachlorobiphenyl (CB-153) and dichlorodiphenyl dichloroethene (p,p'-DDE). Semen characteristics (volume, sperm concentration, total count proportion of progressively motile and morphology) and DNA fragmentation index (DFI) were determined. CAG and GGN repeat lengths were determined by direct sequencing of leukocyte DNA. Results A statistically significant interaction was found between the CB-153 group and CAG repeat category in relation to sperm concentration and total sperm count (P=0.03 and 0.01, respectively). For p,p'-DDE, in the European cohorts a significant interaction was found in relation to DFI (P=0.01). For CAG<20, sperm concentration and total sperm count were 35 and 42% lower, respectively, when the group with CB-153 exposure above median was compared with that below the median. DF1 was 40% higher in the high p,p'-DDE exposure group for CAG < 21. Conclusions This study indicated that the androgen receptor CAG repeat length might modify the susceptibility of an individual to the adverse effects of persistent organohalogen pollutant exposure on semen quality. Other studies regarding this matter are warranted.
2007
- Chromatin Damage and Male Infertility
[Capitolo/Saggio]
D., Sakkas; D., Bizzaro; Manicardi, Gian Carlo
abstract
There is accumulating evidence linking sperm chromatin damage to poor reproductive outcome. The chromatin damage is associated to sperm anomalies that manifest themselves as breaks in the sperm nuclear DNA, aberrant ratios of protamine and histones in the chromatin, and the presence of apoptotic marker proteins in the ejaculated spermatozoa. This chapter examines the mechanisms involved in generating chromatin damage during spermatogenesis in the human, the techniques used to test sperm chromatin and how they may affect reproductive outcome, and how to reduce the risk of using spermatozoa with chromatin damage.
2007
- Effetti dell'esposizione a interferenti endocrini sull'integrità cromatinica/genomica degli spermatozoi umani.
[Capitolo/Saggio]
Stronati, A; Bordicchia, M; Cecati, M; Marcaccio, S; Manicardi, Gian Carlo; Sakkas, D; Spanò, M; Long, M; Kruger, T; Hjielmborg, Ps; Johnsson BO, Ag; Rignell Hydbom, A; Rylander, L; Giwercman, A; Toft, G; Bonde, Jp; Bonefeld Jorgensen, Ec; Bizzaro, D.
abstract
.
2007
- Influenza delle mutazioni del canale del sodio sulla resistenza ai piretroidi in Myzus persicae.
[Abstract in Atti di Convegno]
Mazzoni, E.; Pavesi, F.; Cassanelli, Stefano; Manicardi, Gian Carlo; Cravedi, P.
abstract
Il contributo analizza l'influenza delle mutazioni del canale del sodio sulla resistenza ai piretroidi in diverse popolazioni dell'afide verde del pesco Myzus persicae.
2007
- Molecular and biochemical diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian populations of the peach potato aphid, Myzus persicae (Sulzer).
[Relazione in Atti di Convegno]
Criniti, A; Cassanelli, Stefano; Mazzoni, E; Pavesi, F; Bizzaro, D; Manicardi, Gian Carlo
abstract
The contribution deals with a monitoring of molecular and biochemical diagnostic methods utilised for analysis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian populations of the peach potato aphid, Myzus persicae.
2007
- Presenza di resistenza “kdr” in popolazioni di Musca domestica L. (Diptera, Muscidae) raccolte il provincia di Piacenza.
[Abstract in Atti di Convegno]
Mazzoni, E.; Manicardi, Gian Carlo; Bizzaro, D.
abstract
Il contributo analizza la diffusione di mutazioni nel gene del canale del sodio alla base della resistenza “kdr” in popolazioni di Musca domestica L. (Diptera, Muscidae) raccolte il provincia di Piacenza.
2007
- Relation between serum xenobiotic-induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations
[Articolo su rivista]
M., Long; A., Stronati; D., Bizzaro; T., Kruger; Manicardi, Gian Carlo; P. S., Hjelmborg; M., Spano; A., Giwercman; G., Toft; J. P., Bonde; E. C., Bonefeld Jorgensen
abstract
Persistent organic pollutants (POPs) can interfere with hormone activities and are suspected as endocrine disrupters involved in disorders, e.g. reproductive disorders. We investigated the possible relation between the actual integrated serum xenoestrogenic, xenoandrogenic and aryl hydrocarbon receptor (AhR) activities, and the sperm DNA damage and sperm apoptotic markers of 262 adult males (54 Inuits from Greenland, 69 from Warsaw (Poland), 81 from Sweden, and 58 from Kharkiv (Ukraine)) exposed to different levels of POPs. Xenobiotic-induced receptor activities were determined by receptor-mediated luciferase reporter gene expression. Sperm DNA damage was measured using terminal deoxynucleotidyl transferase-driven dUTP nick labeling assay (TUNEL) and pro- (Fas) and anti-apoptotic (Bcl-xL) markers were determined by immune methods. Different features of xenobiotic-induced receptor activity in serum and sperm DNA fragmentation and apoptotic markers existed between the Inuits and the European Caucasians. Negative correlations between xenobiotic-induced receptor activities and DNA damage were found for Inuits having relatively lower xenoestrogenic, lower dioxin-like activity, and lower sperm DNA damage, but higher xenoandrogenic activity. In contrast, in the European groups, xenobiotic-induced receptor activities were found to be positively correlated with the DNA damage. Further research must elucidate whether altered receptor activities in concerted action with genetic and/or nutrient factors may have protecting effect on sperm DNA damage of the Inuit population.
2007
- Safety of Sperm for use in ICSI
[Capitolo/Saggio]
Sakkas, D; Seli, E; Bizzaro, D; Manicardi, Gian Carlo; Jakab, A; Huszar, G.
abstract
.
2007
- Xenoandrogenic activity in serum differs across European and inuit populations
[Articolo su rivista]
Kruger, T; Hjelmborg, Ps; Jonsson, Ba; Hagmar, L; Giwercman, A; Manicardi, Gian Carlo; Bizzaro, D; Spano, M; Rignell Hydbom, A; Pedersen, Hs; Toft, G; Bonde, Jp; Bonefeld Jorgensen, Ec
abstract
Background: Animal and in vitro studies have indicated that human male reproductive disorders can arise as a result of disrupted androgen receptor (AR) signalling by persistent organic pollutants (POPs) . Our aim in the present study was to compare serum xenoandrogenic activity between study groups with different POP exposures and to evaluate correlations to the POP proxy markers 2,2´,4,4´,5,5´-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl) -ethylene (p,p´-DDE) .Methods: We determined xenoandrogenic activity in the serum fraction containing the lipophilic POPs but free of endogenous hormones. Adult male serum (n = 261) from Greenland, Sweden, Warsaw (Poland) , and Kharkiv (Ukraine) was analyzed. Xenoandrogenic activity was determined as the effect of serum extract alone (XAR) and in the presence of the synthetic AR agonist R1881 (XARcomp) on AR transactivated luciferase activity.Results: The study groups differed significantly with respect to XARcomp activity, which was increased in the Inuits and decreased in the European study groups ; we observed no difference for XAR activity. We found the highest level of the AR antagonist p,p´-DDE in Kharkiv, and accordingly, this study group showed the highest percent of serum samples with decreased XARcomp activities. Furthermore, the percentage of serum samples with decreased XARcomp activities followed the p,p´-DDE serum level for the European study groups. No correlations between serum XAR or XARcomp activities and the two POP markers were revealed.Conclusions: The differences in XARcomp serum activity between the study groups suggest differences in chemical exposure profiles, genetics, and/or lifestyle factors.
2006
- Acetylcholinesterase mutation in an insecticide-resistant population of the codling moth Cydia pomonella (L.)
[Articolo su rivista]
Cassanelli, Stefano; M., Reyes; M., Rault; Manicardi, Gian Carlo; B., Sauphanor
abstract
Two strains of Cydia pomonella (L.) (Lepidoptera: Tortricidae) were selected in the lab by exposure to increasing concentrations of diflubenzuron (Rdfb strain) or azinphos-methyl (Raz strain). Insecticide bioassays showed that the adults of the Rdfb strain exhibited a 2.6-fold and a 7.7-fold resistance ratio to azinphos-methyl and carbaryl, respectively compared to a susceptible strain (S) whereas the adults of the Raz strain exhibited a 6.7-fold resistance ratio to azinphos-methyl and a 130-fold resistance ratio to carbaryl. In the Raz strain, a target site resistance mechanism was suggested by the inhibition of acetylcholinesterase (AChE) activity. In fact the ki values did not discriminate the S and Rdfb strains, while the Raz strain exhibited a 1.7-fold and a 14-fold increase in ki value compared to the S strain for azinphos-methyl oxon and carbaryl, respectively. To verify this hypothesis, two cloned AChE cDNAs sequences (named cydpom-ace2 e cydpom-ace1) were compared between the susceptible and the resistant strains. No difference in the deduced amino acid sequence was found in cydpom-ace2 (orthologous to the Drosophila melanogaster AChE). In the putative eydpom-ace1 (paralogous to the Drosophila AChE), a single amino acid substitution F399V was exclusively present in the Raz strain. The F399 lined the active site of the enzyme and the F399V substitution likely could influence the accessibility of different types of inhibitors to the catalytic site of the insensitive cydpom-ace1. (c) 2006 Elsevier Ltd. All rights reserved.
2006
- Aspetti genetici della resistenza agli insetticidi.
[Articolo su rivista]
Manicardi, Gian Carlo; Cassanelli, Stefano; Criniti, A; Mazzoni, E; Bizzaro, D.
abstract
Suppl. 1 al numero del 12/18 maggio 2006
2006
- Association between exposure to persistent organohalogen pollutants and epididymal and accessory sex gland function: Multicentre study in Inuit and European populations
[Articolo su rivista]
S., Elzanaty; A., Rignell Hydbom; Bag, Jonsson; Hs, Pedersen; Jk, Ludwicki; M., Shevets; V., Zvyezday; G., Toft; Jp, Bonde; L., Rylander; L., Hagmar; E., Bonefeld Jorgensen; M., Spano; D., Bizzaro; Manicardi, Gian Carlo; A., Giwercman
abstract
Exposure to persistent organochlorine pollutants (POPs) may have negative impact on male reproductive function. We, therefore, investigated the association between serum levels of POPs and epididymal and accessory sex gland function. Serum levels of CB-153, pp'-DDE and seminal markers of epididymal [neutral-a glucosidase (NAG)], prostatic [prostate specific-antigen (PSA)] and zinc, and seminal vesicle function (fructose) were measured from 135 Swedish fishermen and fertile men from Greenland (n = 163), Warsaw, Poland (n = 167) and Kharkiv, Ukraine (n = 158). Multiple linear regression analyses, adjusting for potential confounders, were employed using both continuous and categorized exposure variables. Both exposure and outcome variables were log transformed. Considering the consistency between models with either continuous or categorized CB-153 levels, negative associations with the activity of NAG were found among Greenlandic men (mean difference 7.0 mU/ejaculate, 95% CI 3.0, 34), and in the aggregated cohort (mean difference 4.0 mU/ejaculate, 95% Cl -0.2, 8.0). A positive association was observed between CB-153 and PSA as well as zinc among Kharkiv men. In the Swedish cohort, a negative association was found between CB-153 and fructose. In conclusion, the negative effects of POP on sperm motility, observed in the same study population might partly be caused by post-testicular mechanisms, involving a decreased epididymal function. (c) 2006 Elsevier Inc. All rights reserved.
2006
- Dioxin-like activities in serum across European and Inuit populations.
[Articolo su rivista]
Long, M; Andersen, Bs; Lindh, Ch; Hagmar, L; Giwercman, A; Manicardi, Gian Carlo; Bizzaro, D; Span, M; Toft, G; Pedersen, Hs; Zvyezday, V; Bonde, Jp; BONEFELD JORGENSEN, Ec
abstract
Persistent organic pollutants (POPs) such as polychlorinated dibenzo-p-dioxins/furans, polychlorinated biphenyls (PCBs) and organochlorine pesticides can cause a series of adverse effects on e.g. reproduction in animals and humans, many of which involve the aryl hydrocarbon receptor (AhR). The aim of the present study was to compare the integrated serum level of AhR mediated activity among European and Inuit populations, and evaluate whether the activity was associated to the selected POP markers, 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE).
2006
- Genetic variability among different Italian populations of the aphid Myzus persicae
[Articolo su rivista]
A., Criniti; E., Mazzoni; Pecchioni, Nicola; D., Rau; Cassanelli, Stefano; D., Bizzaro; Manicardi, Gian Carlo
abstract
Aphids life cycle includes cyclical or obligate parthenogenesis, therefore low genetic variation is expected in these insects. Genetic diversity in crop pest aphids such as Myzus persicae, is also influenced by the extensive use of insecticides which favoured the selection of few resistant strains. In the present work, 18 M. persicae populations collected in different Italian regions, were studied by RAPD-PCR (Random Amplified Polymorphic DNA) to assess genetic variability among populations. Twelve different random primers, selected out of 24 previously investigated, were employed for genomic DNA amplification. A total of 150 fragments from the aphid M. persicae populations, and 31 fragments in two outgroups were identified. An UPGMA cluster analysis based on NEI and LI's genetic distance revealed that the M. persicae populations could be divided into two groups: aphids from Central and Southern Italy were generally located in the same cluster, while aphids from Northern Italy were more often located in the other. The presence of a positive correlation between genetic and geographical distance suggests that at least a portion of the interpopulation polymorphism evidenced could be caused by restricted gene flow.
2006
- Impact of PCB and p,p'-DDE contaminants on human sperm Y : X chromosome ratio: Studies in three European populations and the inuit population in Greenland
[Articolo su rivista]
T., Tiido; A., Rignell Hydbom; Bag, Jonsson; Yl, Giwercman; Hs, Pedersen; B., Wojtyniak; Jk, Ludwicki; V., Lesovoy; V., Zvyezday; M., Spano; Manicardi, Gian Carlo; D., Bizzaro; EC Bonefeld, Jorgensen; G., Toft; Jp, Bonde; L., Rylander; L., Hagmar; A., Giwercman
abstract
Recent studies indicate that persistent organohalogen pollutants (POPs) may contribute to sex ratio changes in offspring of exposed populations. Our aim in the present study was to investigate whether exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) and dichlorodiphenyldichloroethene (pp'-DDE) affects sperm Y:X chromosome distribution. SUBJECTS AND METHODS: We obtained semen and blood for analysis of PCB-153 and pp'-DDE levels from 547 men from Sweden, Greenland, Poland (Warsaw), and Ukraine (Kharkiv), with regionally different levels of POP exposure. The proportion of Y- and X-chromosome-bearing sperm in the semen samples was determined by two-color fluorescence in situ hybridization analysis. RESULTS: Swedish and Greenlandic men had on average significantly higher proportions of Y sperm (in both cohorts, 51.2%) and correspondingly higher lipid-adjusted concentrations of PCB-153 (260 ng/g and 350 ng/g, respectively) compared with men from Warsaw (50.3% and 22 ng/g) and Kharkiv (50.7% and 54 ng/g). In the Swedish cohort, log-transformed PCB- 153 and log-transformed pp'-DDE variables were significantly positively associated with Y-chromosome fractions (p-values 0.04 and < 0.001, respectively). On the contrary, in the Polish cohort PCB-153 correlated negatively with the proportion of Y-bearing fraction of spermatozoa (p = 0.008). CONCLUSIONS: The present study indicates that POP exposure might be involved in changing the proportion of ejaculated Y-bearing spermatozoa in human populations. Intercountry differences, with different exposure situations and doses, may contribute to varying Y:X chromosome ratios.
2006
- Molecular targeting for insecticide resistance in pest crop insect genomes
[Relazione in Atti di Convegno]
Cassanelli, Stefano; Bizzaro, D; Mazzoni, E; Criniti, A; Manicardi, Gian Carlo
abstract
The contribution illustrate a number of inno9vative methods for molecular targeting of insecticide resistance in pest crop insect genomes.
2006
- RELATIONSHIP BETWEEN CHROMOSOMAL TRANSLOCATION AND FE4 GENE IN THE HOLOCENTRIC CHROMOSOMES OF THE INSECT Myzus persicae
[Relazione in Atti di Convegno]
Criniti, A; Bordicchia, M; Mazzoni, E; Cassanelli, Stefano; Cecati, M; Manicardi, Gian Carlo
abstract
The contribution analyses the presence of a RELATIONSHIP BETWEEN CHROMOSOMAL TRANSLOCATION AND FE4 GENE IN THE HOLOCENTRIC CHROMOSOMES OF THE aphid Myzus persicae
2006
- Relationship between sperm chromatin integrity and dietary exposure to PCBs and DDT in European and Inuit populations
[Abstract in Rivista]
Spano, M; Toft, G; Hagmar, L; Eleuteri, P; Cordelli, E; Leter, G; Rescia, M; Villani, P; Jonsson, Bag; Rignell Hydbom, A; Rylander, L; Giwercman, A; Pedersen, Hs; Bonefeld Jorgensen, Ec; Manicardi, Gian Carlo; Bizzaro, D; Ludwicki, Jk; Zvyezday, V; Bonde, Jp
abstract
.
2006
- Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p '-DDE in European and Inuit populations
[Articolo su rivista]
Stronati, A; Manicardi, Gian Carlo; Cecati, M; Bordicchia, M; Ferrante, L; Spano, M; Toft, G; Bonde, Jp; Jonsson, Bag; Rignell Hydbom, A; Rylander, L; Giwercman, A; Pedersen, Hs; Bonefeld Jorgensen, Ec; Ludwicki, Jk; Lesovoy, V; Sakkas, D; Bizzaro, D.
abstract
Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting proapoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to he investigated in order to fully assess the population variations observed.
2006
- Reproductive hormone levels in men exposed to persistent organohalogen pollutants: A study of Inuit and three European cohorts
[Articolo su rivista]
A., Giwercman; A., Rignell Hydbom; G., Toft; L., Rylander; L., Hagmar; C., Lindh; Hs, Pedersen; Jk, Ludwicki; V., Lesovoy; M., Shvets; M., Spano; Manicardi, Gian Carlo; D., Bizzaro; EC Bonefeld, Jorgensen; Jp, Bonde
abstract
OBJECTIVE: Persistent organohalogen pollutant (POP) exposure may have a negative impact on reproductive function. The objective of this study was to assess the impact of POP exposure on the male hypothalamo-pituitary-gonadal axis. PARTICIPANTS: Participants included 184 Swedish fishermen and spouses of pregnant women from Greenland (n = 258), Warsaw, Poland (n = 113), and Kharkiv, Ukraine (n = 194). EVALUATIONS/MEASUREMENTS: Serum levels of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and dichlorodiphenyl dichloroethene (p,p'-DDE) were determined in the four populations, showing different exposure patterns: Swedish fishermen, high CB-153/low p,p'-DDE; Greenland, high CB-153/high p,p'-DDE; Warsaw, low CB-153/moderate p,p'-DDE; Kharkiv, low CB-153/high p,p'-DDE. Serum was also analyzed for testosterone, estradiol sex hormone-binding globulin (SHBG), inhibin B, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Free testosterone levels were calculated based on testosterone and SHBG. RESULTS: We found significant center-to-center variations in the associations between exposure and the outcomes. The most pronounced effects were observed in Kharkiv, where statistically significant positive associations were found between the levels of both CB-153 and p,p'-DDE and SHBG, as well as LH. In Greenland, there was a positive association between CB-153 exposure and LH. In the pooled data set from all four centers, there was positive association between p,p'-DDE and FSH levels [beta = 1.1 IU/L; 95% confidence interval (CI), 1.0-1.1 IU/L]. The association between CB-153 levels and SHBG was of borderline statistical significance (beta = 0.90 nmol/L; 95% CI, -0.04 to 1.9 nmol/L). CONCLUSIONS: Gonadotropin levels and SHBG seem to be affected by POP exposure, but the pattern of endocrine response is the subject of considerable geographic variation.
2006
- Semen quality and exposure to persistent organochlorine pollutants
[Articolo su rivista]
G., Toft; A., Rignell Hydbom; E., Tyrkiel; M., Shvets; A., Giwercman; Ch, Lindh; Hs, Pedersen; Jk, Ludwicki; K., Lesovoy; L., Hagmar; M., Spano; Manicardi, Gian Carlo; EC Bonefeld, Jorgensen; Am, Thulstrup; Jp, Bonde
abstract
Background: Inconsistent results have been found in previous human studies on male reproductive toxicity of persistent organochlorine pollutants. The majority of studies have been conducted among selected populations of infertility clients or among occupational cohorts including a limited number of participants. Methods: We conducted a cross-sectional study of semen quality and serum concentration of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE) among 763 men. We included men from all regions in Greenland (n = 194), fishermen from Sweden (n = 185), inhabitants of the city of Kharkiv, Ukraine (n = 195), and inhabitants of the city of Warsaw, Poland (n = 189). Blood samples were analyzed for CB-153 and p,p'-DDE using gas chromatography-mass spectrometry and adjusted for serum lipids. Results: Sperm concentration was not impaired with increasing serum CB-153 or p,p'-DDE levels in any of the separate groups or overall. Similarly, the proportion of morphologically normal sperm was not associated with either CB-153 or p,p'-DDE blood concentration. However, sperm motility was inversely related to CB-153 concentration in Greenland and the Swedish fishermen population. Across all 4 regions, the sperm motility decreased on average by 3.6% (95% confidence interval = 1.7% to 5.6%) per one-unit increase in the log of blood CB-153 (ng/g lipid). The concentration of p,p'-DDE was negatively associated with sperm motility in the Greenlandic population and in the compiled dataset. Conclusion: Adult exposure to persistent organochlorine pollutants within the ranges observed in the present study is not likely to cause reduction in sperm concentration or morphology. However, higher exposure may be associated with impaired sperm motility.
2006
- Seminal apoptosis and serum levels of CB153 and DDE in European and Inuit populations
[Abstract in Rivista]
Stronati, A; Bizzaro, D; Cecati, M; Bordicchia, M; Ferrante, L; Spano, M; Toft, G; Bonde, Jp; Hagmar, L; Jonsson, B; Rignell Hydbom, A; Rylander, L; Giwercman, A; Pedersen, Hs; Bonefeld Jorgensen, Ec; Ludwicki, Jk; Zvyezday, V; Sakkas, D; Manicardi, Gian Carlo
abstract
.
2006
- Sostanze esogene ad azione ormono simile e markers di apoptosi in spermatozoi umani maturi
[Capitolo/Saggio]
Stronati, A; Bordicchia, M; Cecati, M; Spano, M; BONEFELD JORGENSEN, Ec; Toft, G; Bonde, Jp; Hagmar, L; JONSSON BO, Ag; RIGNELL HYDBOM, A; Giwercman, A; Pedersen, Hs; Ludwicki, Jk; Zvyezday, V; Sakkas, D; Manicardi, Gian Carlo; Bizzaro, D.
abstract
L'articolo analizza le relazioni esistenti tra sostanze esogene ad azione ormono simile e markers di apoptosi in spermatozoi umani maturi provenienti da 4 popolazioni europee (Progetto INUENDO)
2006
- Xenobiotic Activities in Serum of Inuit and European Populations: Effects on Semen DNA Quality Markers
[Abstract in Rivista]
Bonefeld Jorgensen, E. C*; Bizzaro, D†; Manicardi, Gian Carlo; Krüger, T*; Long, M*; Hjelmborg, P. S*; Stronati, A†; Giwercman, A§; Hagmar, L¶; Ludwicki, J. K∥; Zvyezday, V#; Toft, G**; Bonde, J. P**; Spanò, M††
abstract
Human exposure to environmental contaminants is ubiquitous and can affect individuals living close to as well as remote from the sources of contaminants. Persistent organic pollutants (POPs) include polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and certain pesticide residues. Exposure to POPs elicits a number of species- and tissue-specific toxic responses including effects on the reproductive-, immune- and thyroid system. Adverse effects have been documented in wildlife populations, whereas no clear cut evidence for adverse endocrine-related human health effects has been obtained. The toxicological assessment of individuals is complicated by the life long exposure to a complex mixture of contaminants. The present study compare the actual serum level of xeno-hormone (estrogenic and androgenic) and dioxin-like activity between populations from Greenland (Inuit's) and Europe (Sweden, Poland, Ukraine) for finally to evaluate possible associations to the POP markers PCB153 and p,p'-DDE, semen DNA quality markers and lifestyle parameters.The SPE-HPLC serum fraction containing the actual mixture of bio-accumulated POPs, free of endogenous hormones, was used to determine the effect on the estrogen- (ER) and androgen-receptor (AR) trans-activity in stable transfected ER-luc MVLN and transient transfected ARE-luc CHO-K1 cells, respectively. Serum dioxin-like activity was determined in serum fat extracts using the stable AhR-luc transfected Hepa 1.12cR cells. Sperm DNA damage was measured using Terminal Deoxynucleotydil Transferase assay and apoptotic markers Fas and Bcl-xL were determined by immune methods. Fluoremetric sperm chromatin structure assay was used to assess sperm DNA/chromatin integrity (DFI).The integrated net xenobiotic activities determined in serum elicited a district dependent pattern. Compared to controls significantly effects of the actual serum POP fraction on the activity of each of the receptors (ER, AR and AhR) was observed. Inuit's differed from the European groups by a higher frequency of samples with antagonized ER, higher AR potency and lower dioxin-like activity. Multiple regressions showed an interaction between semen DNA quality markers (DFI, DNA damage) and xenoestrogenic and dioxin-like activity across the groups. In the separate study groups Inuit's showed a negative correlation between the DNA quality markers and ER and AhR activities and no or few positive correlations for the European samples. No consistent association to the two POP markers was found.The data suggested that different POP burden profiles elicit differences in net xenobiotic receptor activities and effects on semen DNA quality markers. The Inuit's in general differed from the European groups. Future studies might elucidate whether genetic and/or life style factors play a role in the observed differences.
2006
- Xenoestrogenic activity in blood of European and Inuit populations.
[Articolo su rivista]
BONEFELD JORGENSEN, E; Hjelmborg, Ps; Reiner, Ts; Andersen, Bs; Lesovoy, V; Lindh, Ch; Hagmar, L; Giwercman, A; Erlandsen, M; Manicardi, Gian Carlo; Span, M; Toft, G; Bonde, Jp
abstract
Human exposure to persistent organic pollutants (POPs) is ubiquitous and found in all individuals. Studies have documented endocrine disrupting effects and impact on reproduction. The aim of the present study was to compare the level of xenoestrogenic activity in serum of groups with varying POP exposure, and to evaluate correlations to the POP biomarkers, 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE). No strong consistent association between xenoestrogenic net activity and the two POP markers was found. The results showed that the selected POP markers alone can not predict the integrated xenoestrogenic serum activity. Correlations to the POP markers were found at the extreme edge; the Inuit's and Warsaw study groups eliciting high frequency of samples with ER antagonistic and agonistic activity, respectively. We suggest that the variation in xenoestrogenic serum activity reflects differences in POP exposure mixture, genetic factors and/or life style factors.
2005
- Fertility in four regions spanning large contrasts in serum levels of widespread persistent organochlorines: A cross-sectional study
[Articolo su rivista]
Gunnar, Toft; Anna, Axmon; Aleksander, Giwercman; ANE MARIE, Thulstrup; ANNA RIGNELL, Hydbom; HENNING SLOTH, Pedersen; JAN K., Ludwicki; Valentina, Zvyezday; Andery, Zinchuk; Marcello, Spano; Manicardi, Gian Carlo; EVA C., BONEFELD JRGENSEN; Lars, Hagmar; JENS PETER BONDE AND, Inuendo
abstract
BACKGROUND: Persistent organochlorine pollutants (POPs) may interfere with reproductive function but direct evidence in humans is very limited. METHODS: Fertility was examined in four regions with contrasting blood levels of POPs. Pregnant women and their partners in Warsaw (Poland), Kharkiv (Ukraine) and Greenland were consecutively enrolled during antenatal visits. Swedish fishermen and their spouses were recruited separately and independently of current pregnancy. Lipid adjusted serum concentrations of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (DDE) were available for both partners. Time to pregnancy interviews were obtained among 2269 women and 798 men provided a semen sample. RESULTS: Inuits had high levels of both POP markers, Swedish fishermen were high in CB-153 but low in DDE, men from Kharkiv were high in DDE and low in CB-153 while men from Warsaw were low in CB-153 and had intermediate DDE levels. Compared to Warsaw couples, fecundability was reduced among couples from Kharkiv [adjusted fecundability ratio (FR) 0.64 (95% CI 0.5-0.8)] and elevated in Swedish fishermen families [FR 1.26 (95% CI 1.0-1.6)]. Adjusted geometric means of sperm counts and morphology did not differ between regions while sperm motility was higher in men living in Warsaw. CONCLUSION: We observed regional differences in time to pregnancy and sperm motility that may be related to regional differences in POP blood levels, but other interpretations are also plausible. In particular, differences in access to safe contraception and in the prevalence of contraceptive failures are most likely to bias comparisons of time to pregnancy.
2005
- Il Pianeta Acqua nel Continente Agricoltura (Congresso Nazionale dell’Associazione Italiana delle Società Scientifiche Agrarie – AISSA)
[Altro]
Stanca, Am; Arru, Laura; Bignami, Cristina; Conte, Angela; Endrighi, Emiro; Franchini, ; Lofiego, D; Manicardi, Gian Carlo; Orlandini, Stefano; Pellegrini, ; Ulrici, Alessandro; Bacarella, Borin; Dazzi, Espen; Gallerani, Giupponi; Magnani, ; Pecchioni, Nicola; Poni, Rossi; Zanni,
abstract
Nell’era della specializzazione nel settore della Ricerca Scientifica, il Convegno ha l’ambizione di mettere insieme i singoli componenti del mondo scientifico agrario, di farli interagire tra di loro e di tentare di affrontare il problema Acqua in modo interdisciplinare. L’avanzamento delle conoscenze sul ruolo dell’acqua nel “Continente Agricoltura” garantirà ricadute di notevole interesse a breve, medio e lungo termine, per migliorare ulteriormente l’interazione “Organismi viventi di interesse agrario e forestale - Terreno – Atmosfera”. L’obiettivo finale è infatti quello di assicurare per il futuro uno sviluppo sostenibile, grazie alla razionale gestione di un fattore ambientale ed economico primario, l’Acqua.
2005
- Inter-population variations in concentrations, determinants of and correlations between 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE): A cross-sectional study of 3161 men and women from Inuit and European populations
[Articolo su rivista]
JONSSON BO, Ag; Rylander, L; Lindh, C; RIGNELL HYDBOM, A; Giwercman, A; Toft, G; Pedersen, Hs; Ludwicki, Jk; Goralczyk, K; Zvyezday, V; Spano, M; Bizzaro, D; BONEFELD JORGENSEN, Ec; Manicardi, Gian Carlo; BONDE JP AND HAGMAR, L.
abstract
The study is part of a collaborative project (Inuendo), aiming to assess the impact of dietary persistent organochlorine pollutants (POPs) on human fertility. The aims with the present study are to analyze inter-population variations in serum concentrations of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE), to assess inter-population variations in biomarker correlations, and to evaluate the relative impact of different determinants for the inter-individual variations in POP-biomarkers. CB-153 concentrations were much higher in Inuits and Swedish fishermen's populations than in the populations from Eastern Europe, whereas the pattern was different for p,p'-DDE showing highest concentrations in the Kharkiv population. The correlations between the POP-biomarkers varied considerably between the populations, underlining that exposure sources differ and that the choice of representative biomarkers of overall POP exposure has to be based on an analysis of the specific exposure situation for each population. Age and gender were consistent determinants of serum POPs; seafood was of importance only in the Inuit and Swedish populations.
2005
- Relationship among expression, amplification, and methylation of FE4 esterase genes in Italian populations of Myzus persicae (Sulzer) (Homoptera : Aphididae)
[Articolo su rivista]
D., Bizzaro; E., Mazzoni; E., Barbolini; S., Giannini; Cassanelli, Stefano; F., Pavesi; P., Cravedi; Manicardi, Gian Carlo
abstract
The wide use of insecticides containing an esteric group selected resistant Myzus persicae populations characterised by the overproduction of one of two closely related carboxylesterases (E4 and FE4). In this paper, we present data collected from Italian population indicating that all the 22 populations analysed possess amplified FE4 gene only. The estimation of FE4 copy number, carried out by densitometric scanning of dot and Southern blots, puts in evidence that the different populations possess a gene copy number ranging from 6 to 104. Statistical analysis shows the existence of a high positive correlation between gene copy number and total esterase activity. In aphid strain with low FE4 copy number, these genes are almost totally methylated. On the contrary, aphid strains with high FE4 gene number evidenced highly variable methylation levels and absence of correlation between the number of genes and their methylation state. The same result has been observed when comparing FE4 methylation levels and esterase activity.
2005
- The significance of sperm nuclear DNA strand breaks on reproductive outcome
[Articolo su rivista]
M., Spano; E., Seli; D., Bizzaro; Manicardi, Gian Carlo; D., Sakkas
abstract
Purpose of review A growing body of evidence indicates that ejaculated spermatozoa from men being treated with intracytoplasmic sperm injection contain nuclear abnormalities. Many of these nuclear anomalies manifest themselves as breaks in the sperm nuclear DNA. This review examines the mechanisms involved in generating DNA strand breaks during spermatogenesis in the human, the main techniques used to assess the sperm nucleus and the evidence, in relation to assisted reproduction, showing that sperm nuclear DNA strand breaks may impact on reproductive outcome. Recent findings Techniques such as the TUNEL assay and the sperm chromatin structure assay both show increased levels of DNA abnormalities in spermatozoa from men who have poor semen parameters. The reproductive parameters affected by an increased presence of DNA abnormalities in ejaculated spermatozoa include! fertilization, blastocyst development, and pregnancy rates. Summary There is accumulating evidence linking sperm nuclear DNA anomalies to poor reproductive outcome in relation to assisted reproduction technologies. The tests currently available only provide an inkling of the impact of sperm nuclear DNA abnormalities on reproductive outcomes. Although the impact an abnormal paternal genome may have on reproductive outcome is unquestionably less than that of its female counterpart, it cannot be ignored.
2005
- Use of the RFLP-PCR diagnostic test for characterizing MACE and kdr insecticide resistance in the peach potato aphid Myzus persicae
[Articolo su rivista]
Cassanelli, Stefano; B., Cerchiari; S., Giannini; D., Bizzaro; E., Mazzoni; Manicardi, Gian Carlo
abstract
The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids.
2005
- X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid Aphis pomi
[Articolo su rivista]
A., Criniti; G., Simonazzi; Cassanelli, Stefano; M., Ferrari; D., Bizzaro; Manicardi, Gian Carlo
abstract
Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA(3) staining reveals that these silver positive telomeres are, the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.
2004
- Human cervical mucus can act in vitro as a selective barrier against spermatozoa carrying fragmented DNA and chromatin structural abnormalities
[Articolo su rivista]
Bianchi, Giampaolo; A., De Agostini; J., Fournier; C., Guidetti; N., Tarozzi; D., Bizzaro; Manicardi, Gian Carlo
abstract
Purpose: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. Methods: Sperm chromatin abnormalities have been evaluated by Chromomycin A(3) and endogenous nick translation. Results: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. Conclusions: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.
2004
- Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation
[Articolo su rivista]
Jm, Morrell; O., Moffatt; D., Sakkas; Manicardi, Gian Carlo; D., Bizzaro; M., Tomlinson; H., Nilsson; Pv, Holmes
abstract
Purpose: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. Methods: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm. density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. Results: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. Conclusions: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.
2004
- Rimodellamento nucleare e apoptosi nelle oligo-asteno-teratozoospermie.
[Capitolo/Saggio]
D., Bizzaro; Manicardi, Gian Carlo; N., Tarozzi; A., Stronati; M., Bordicchia; M., Cecati; E., Seli; D., Sakkas
abstract
L'articolo analizza le relazioni esistenti tra modificazioni della cromatina e apoptosi in spermatozoi provenienti da soggetti affetti da oligo-asteno-teratozoospermie.
2004
- The presence of abnormal spermatozoa in the ejaculate: did apoptosis fail?
[Articolo su rivista]
Sakkas, D; Seli, E; Manicardi, Gian Carlo; Nijs, M; Ombelet, W; Bizzaro, D.
abstract
With the successful use of Assisted Reproduction, in particular intracytoplasmic sperm injection (ICSI), to treat infertile couples we have become less discriminating with the quality of spermatozoa we use to treat our patients. Numerous studies have shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities in their nuclear DNA is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the packaging of the chromatin during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve our knowledge about certain causes of male infertility. More importantly, the impact of such sperm, if selected to perform ICSI, needs to be better understood so that any detrimental paternal effects can be avoided.
2003
- Abnormal spermatozoa in the ejaculate: Abortive apoptosis and faulty nuclear remodelling during spermatogenesis
[Articolo su rivista]
Sakkas, D; Seli, E; Bizzaro, D; Tarozzi, N; Manicardi, Gian Carlo
abstract
The mechanisms responsible for producing abnormal spermatozoa in the ejaculate are relatively unknown. Numerous studies have now shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa and the abnormal persistence of apoptotic marker proteins. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the replacement of histones with protamines during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve knowledge about certain causes of male infertility.
2003
- Analysis of insect holocentric chromosomes by atomic force microscopy
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
In order to go in depth into the analysis of holocentric chromosome structure, atomic force microscopy (AFM) was applied to metaphase plates of the aphid Megoura viciae. AFM showed that aphid chromatids adhere to one another without any prominent structure detectable between them and without any evidence of chromosomal constrictions. AFM thus provided new and reliable evidences at a nanomolecular level concerning the holocentric structure of aphid chromosomes, without any of the artefacts due to sample staining or coating that are usually associated with electron microscopy.
2003
- Cytogenetic analysis on the holocentric chromosomes of the cabbage aphid Brevicoryne brassicae
[Articolo su rivista]
Manicardi, Gian Carlo; Mandrioli, Mauro
abstract
Chromatin organization in the holocentric chromosomes of the aphid Brevicoryne brassicae has been investigated at a cytological level after C-banding, NOR, Giemsa, DAPI and CMA(3) staining. C-banding technique showed the presence of heterochromatic bands on both telomeres of the two X chromosomes, whereas only the longest pair of autosomes show distinct intercalary C-positive bands. Moreover, silver staining and fluorescent in situ hybridization (FISH) with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; these are the only brightly fluorescent C-positive regions revealed after CMA(3) staining, whereas all other heterochromatic bands are DAPI positive.
2003
- Cytogenetic and molecular characterization of the MBSAT1 satellite DNA in holokinetic chromosomes of the cabbage moth, Mamestra brassicae (Lepidoptera)
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo; F., Marec
abstract
Digestion of Mamestra brassicae DNA with DraI produced a prominent fragment of approximately 200 by and a ladder of electrophoretic bands with molecular weights which are a multiple of 200 bp. Southern blotting revealed that this ladder is composed of DNA fragments that are multimers of the 200-by DraI band suggesting that DraI isolated a satellite that has been called Mamestra brassicae satellite DNA 1 (MBSAT1). MBSAT1 is the first satellite DNA isolated in Lepidoptera. In-situ DraI digestion of chromosome spreads, together with fluorescent in-situ hybridization, showed that MBSAT1 sequences are clustered in heterochromatin of the sex chromosomes, Z and W. MBSAT1 was 234 bp long with an AT content of 60.7%. The curvature-propensity plot suggested a curvature in the MBSAT1 structure.
2003
- Sperm nuclear DNA damage in the human
[Relazione in Atti di Convegno]
Sakkas, D; Manicardi, Gian Carlo; Bizzaro, D.
abstract
Review. No abstract available.
2003
- The interrelationships of the Gastrotricha using nuclear small rRNA subunit sequence data, with an interpretation based on morphology
[Articolo su rivista]
Todaro, Mary Antonio Donatello; Littlewood, D. T. J.; Balsamo, M.; Herniou, E. A.; Cassanelli, Stefano; Manicardi, Gian Carlo; Wirz, A.; Tongiorgi, Paolo
abstract
Gastrotrichs are meiobenthic invertebrates of obscure origin and unclear phylogenetic alliances. Uncertainties also plague the intra-group relationship with major contrasts between the evolutionary scenarios inferred from morphology or molecules. In this study we analysed partial sequences of the 18S rDNA gene of 18 taxa (14 new and 4 published) to test morphological estimates of gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data are due to poor sampling. Data were analysed using both maximum parsimony and maximum likelihood. MP topology was then forced to reflect published morphological estimates and the most parsimonious solutions from each constraint analysis was statistically compared against the unconstrained Solution. MP analysis yielded a single tree with few nodes well supported by bootstrap resampling These included the monophyly of the Chaetonotidae and the internal relationships of the members of this family, with Aspidiophorus appearing as the most basal member. The monophyly of the Turbanellidae was also well supported with some suggestion that its sister group might be Mesodasys. Lepidodasyidae was found to be an unnatural taxon with Lepidodasys forming a separated clade but unrelated also to the Thaumastodermatidae. With the exception of genera Lepidodasys and Neodasys, the Macrodasyida appeared to be resolved separately from the Chaetonotida, and Dactylopodola was resolved as the most basal macrodasyid. ML analysis yielded a tree not too dissimilar from MP, although Doctylopodola and Xenodasys were resolved as a clade. Statistics indicate that the Output from Our MP analysis is compatible with the classical view placing representatives of the two orders within two distinct evolutionary lines. Most of the constrained solutions, except the shortest, corroborate the monophyly of the two orders. whereas all five constrained solutions support also the notion that sees Neodasys as an early divergent clade along the Chaetonotida branch. Thus, results are generally compatible with the hypothesised evolutionary scenario based on morphological data, but are in contrast with previous findings from molecules. Future research should consider using the complete SSU rDNA gene sequence in their analysis and additional genes for deeper resolution.
2002
- Cytogenetic and Molecular Analysis of Heterochromatic Areas in the Holocentric Chromosomes of Different Aphid species.
[Capitolo/Saggio]
Manicardi, Gian Carlo; Mandrioli, Mauro; Bizzaro, D.; Bianchi, U.
abstract
L'articolo presenta una revisione delle ricerche di citogenetica classica e molecolare svolte allo scopo di mettere in evidenza le peculiarità dei cromosomi olocentrici degli afidi.
2002
- Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis
[Articolo su rivista]
D., Sakkas; O., Moffatt; Manicardi, Gian Carlo; E., Mariethoz; N., Tarozzi; D., Bizzaro
abstract
Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase 1. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.
2001
- Cytogenetic and molecular analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes)
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo
abstract
In view of their compact genome, pufferfish (Tetraodontiformes) have been proposed as model animal for the study of the vertebrate genome. Despite such interest, cytogenetic information about puffers is still scanty. To fill this gap, a cytogenetic analysis of T. fluviatilis has been performed using both classical and molecular techniques. C-banding, followed by DAPI staining, evidenced that in T. fluviatilis, like all other puffer species so far examined, heterochromatin is essentially AT-rich and it is located at centromeres, whereas staining with CMA(3), silver staining and FISH with a 28S ribosomal RNA gene DNA probe showed 2-4 nucleolar organizing regions (NORs) located in heterochromatic regions in the considered puffer species. FISH with the 5S probe put in evidence both in T. fluviatilis and in T. nigroviridis only a 5S cluster per haploid genome that is physically unlinked with the major ribosomal RNA genes including the 28S rRNA genes. Hybridization with the (TTAGGG)(n) probe showed in all the puffers brightly fluorescent signals uniform both in size and intensity at the end of all the chromosomes. Finally, mariner-like elements (MLEs) have been identified in T. fluviatilis and they have located into the NOR-associated heterochromatin.
2001
- Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception
[Articolo su rivista]
Mj, Tomlinson; O., Moffatt; Manicardi, Gian Carlo; D., Bizzaro; M., Afnan; D., Sakkas
abstract
BACKGROUND: With an increase in the use of assisted reproduction technologies the requirements of the diagnostic semen analysis are constantly changing. METHODS: Spermatozoa from patients undergoing IVF were analysed by examining the conventional semen parameters and DNA/chromatin integrity, using in-situ nick translation (NT) and the Chromomycin A(3) fluorochrome, which indirectly demonstrates a decreased presence of protamine. Samples were examined before and after preparation using discontinuous density gradient centrifugation. RESULTS: Density gradient centrifugation enriched samples by improving the percentage of morphologically normal forms by 138% and sperm nuclear integrity by 450%. Sperm nuclear integrity as assessed by in-situ nick translation (NT) demonstrated a very clear relationship with sperm concentration, motility and morphology. Morphology correlated with fertilization rates of patients undergoing IVF, while NT values of the spermatozoa post-preparation were significantly lower in pregnant patients. CONCLUSIONS: We have demonstrated that along with the classical semen parameters, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating patients to specific assisted reproduction treatments.
2001
- Paternal effects on fertilization, embryo development and pregnancy outcome.
[Relazione in Atti di Convegno]
Sakkas, D.; Manicardi, Gian Carlo; Bizzaro, D.; Moffatt, O.; Tomlinson, M.
abstract
The paper deals with the analyses of paternal effects on fertilization, embryo development and pregnancy outcome.
2000
- Chromosome analysis and molecular characterization of highly repeated DNAs in the aphid Acyrthosiphon pisum (Aphididae, Hemiptera)
[Articolo su rivista]
D., Bizzaro; Mandrioli, Mauro; M., Zanotti; M., Giusti; Manicardi, Gian Carlo
abstract
Despite the interest in aphid biology, information on chromatin organization of their holocentric chromosomes is still limited to few species. In order to fill this gap, we have performed an extensive survey on pea aphid mitotic chromosomes using both classical and molecular cytogenetic techniques. Our results after silver, CMA(3) and DAPI-staining, C-banding and fluorescent in situ hybridization (FISH) using 28S rDNA and 5S rDNA as probes evidenced a tendency of repetitive DNAs to be concentrated on the X chromosomes. FISH experiments with the telomeric probe (TTAGG)(n) revealed bright hybridization signals on each telomere of all Acyrthosiphon pisum chromosomes. No interstitial signals were seen.
2000
- Cytogenetic analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes)
[Articolo su rivista]
Mandrioli, Mauro; Cuoghi, Barbara; Marini, Milena; Manicardi, Gian Carlo
abstract
Because of their compact genome, pufferfish (Tetraodontiformes) have been proposed as a model for the study of the vertebrate genome. The genome of pufferfish is peculiar as it has the structural complexity of the genomes of higher vertebrates, but has small introns and lacks large clusters of highly repetitive sequences. Despite such interest, information about the genetics of pufferfish is still scanty. To fill this gap, we have performed a cytogenetic analysis of the pufferfish, Tetraodon fluviatilis, which can be maintained in an aquarium for a long time and, unlike the pufferfish, Fugu rubripes, it is not difficult to obtain. Karyotype analysis shows that T. fluviatilis has 2n = 42 with two metacentric chromosomes, four submetacentrics, two subtelocentrics and 34 acrocentrics. C-banding, followed by DAPI staining, showed that heterochromatin is essentially AT-rich and is located at centromeres. Staining of the same metaphase plates with CMA(3) showed the presence of four heterochromatic regions located on two pairs of submetacentric chromosomes. Silver staining and FISH with a 28S rDNA probe showed that these GC-rich regions are nucleolar organizing regions (NORs). Finally, regardless of the technique used, no difference in the chromosome complement was found between males and females.
2000
- Molecular and cytogenetic analysis of the goby Gobius niger (Teleostei, Gobiidae)
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo; Machella, N; Caputo, V.
abstract
A molecular cytogenetic study of Gobius niger has been conducted by treating its mitotic chromosomes with silver-, CMA(3)- and DAPI-staining and fluorescent in situ hybridization using four multicopy or repetitive DNAs (the 28S and 5S rDNAs, the TTAGGG telomeric repeat and the mariner-like elements) as probes. In particular, the study proved the presence of NOR heteromorphism and suggested the possible role of the transposable element mariner in its genesis. In situ hybridization with the 5S rDNA probe proved the presence of just one 5S-bringing chromosome pair, whereas hybridization with the telomeric repeat revealed small bright hybridization spots, uniform in size and intensity, on each telomere of all chromosomes but no interstitial signals were noticed.
2000
- Possible consequences of performing intracytoplasmic sperm injection (ICSI) with sperm possessing nuclear DNA damage
[Articolo su rivista]
Sakkas, D.; Manicardi, Gian Carlo; Bizzaro, D.; Bianchi, P. G.
abstract
A number of studies have confirmed that sperm with damaged nuclear DNA are present in human ejaculate. It appears that these sperm are more likely to occur in men with low concentrations of sperm, or poor sperm motility or morphology. In assisted reproductive techniques, in particular intracytoplasmic sperm injection (ICSI), there is a higher statistical chance that sperm possessing damaged DNA will be selected and used to fertilize oocytes. In light of this observation, the question of whether human sperm with damaged DNA can impair fertilization and embryo development is examined.
2000
- Relationship between expression, amplification and methylation of FE4 gene in Myzus persicae (Homoptera, Aphididae)
[Relazione in Atti di Convegno]
Bizzaro, D.; Barbolini, E.; Mazzoni, E.; Mandrioli, Mauro; Manicardi, Gian Carlo; Cravedi, P.; Crema, R.; Pagliai, A. M.; Bianchi, U.
abstract
The paper deals witha precise description of the elationship between expression, amplification and methylation of FE4 gene in Myzus persicae (Homoptera, Aphididae).
2000
- Sperm decondensation during fertilization in the mouse: presence of DNaseI hypersensitive sites in situ and a putative role for topoisomerase II.
[Articolo su rivista]
Bizzaro, D; Manicardi, Gian Carlo; Bianchi, P. G.; Sakkas, D.
abstract
In this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 mu M teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated.
2000
- The use of two density gradient centrifugation techniques and swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies
[Articolo su rivista]
Sakkas, D.; Manicardi, Gian Carlo; Tomlinson, M.; Bizzaro, D.; Mandrioli, Mauro; Bianchi, P. G.; Bianchi, U.
abstract
Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm(R), Percoll(R) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm(R) or Percoll(R) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm(R); n = 39 for Percoll(R)). Spermatozoa prepared using PureSperm(R) (n = 35) and Percoll(R) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm(R) and Percoll(R) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm(R) and Percoll(R) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.
1999
- Cytogenetic analysis of the holocentric chromosomes of the aphid Schizaphis graminum
[Articolo su rivista]
Mandrioli, Mauro; Ganassi, S; Bizzaro, D; Manicardi, Gian Carlo
abstract
Chromatin organization in the holocentric chromosomes of the aphid Schizaphis graminum has been investigated at a cytological level after C-banding, NOR, Giemsa, DAPI and CMA(3) staining. C-banding technique showed the presence of numerous C bands on the two X chromosomes both in telomeric and intercalary regions, whereas autosomes show a small number of heterochromatic bands. Contrary to the results with other aphid species, in S. graminum the C-banding pattern is peculiar to each chromosome pair, thus allowing the identification of homologues and the reliable reconstruction of a karyotype. These cytogenetic data could be useful for the identification of chromosomal rearrangement eventually occurred between different S. graminum biotypes. Moreover, silver staining and fluorescent in situ hybridization (FISH) with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; these are the only brightly fluorescent C-positive regions revealed after CMA(3) staining, whereas all other heterochromatic bands are DAPI positive.
1999
- Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae
[Articolo su rivista]
Mandrioli, Mauro; D., Bizzaro; Manicardi, Gian Carlo; D., Gionghi; L., Bassoli; Bianchi, Umberto
abstract
Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands, located on autosomes, consisting of Haem repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%-94%). A similarity plot showed that sequences were particularly variable in the 50-100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important.
1999
- Cytogenetic characterization of the holocentric chromosomes in the aphids Myzus varians and Myzus cerasi
[Articolo su rivista]
Bizzaro, D; Barbolini, E; Mandrioli, Mauro; Mazzoni, E; Manicardi, Gian Carlo
abstract
Cytogenetic investigations on the holocentric chromosomes of Myzus varians and Myzus cerasi have been carried out using silver staining and C-banding followed by chromomycin A(3) (CMA(3)) and DAPI staining in order to improve our knowledge of these pest crop species and facilitate their identification. In M. varians C-banding pattern is peculiar of each chromosome pair thus allowing the identification of all homologues and the reconstruction of a reliable karyotype whereas in M. cerasi, C-positive regions result to be limited to both the telomeric regions of the X chromosomes. In both species, silver staining localizes rDNA genes on one telomere of each X chromosome; such telomeres are the unique brightly fluorescent C-positive regions after CMA(3) staining, whereas all other heterochromatic bands result DAPI positive.
1999
- High values of DNA content in the hypotalamic neurons of Lophius piscatorius and Diodon holacanthus (Osteichthyes)
[Articolo su rivista]
Benedetti, I; Sassi, D; Mescoli, G; Manicardi, Gian Carlo
abstract
Computerised image analysis on Feulgen-stained preparations indicated that in both Lophius piscatorius and Diodon holacanthus the Feulgen-DNA content of the hypothalamic magnocellular neurons in the preoptic and tuberal complexes largely exceeded 2C. Subsequent quantitative microfluorometric evaluation revealed that the DNA content of these cells was as higher as 68C in L. piscatorius and 84C in D. holacanthus. The presence of high levels of DNA content in neuron nuclei is well known in the case of the giant neurons of Molluscs but, although it has recently been found in the supramedullary neuron cluster of the same Teleosts used in this study, it must be considered exceptional for the nervous system of Vertebrates.
1999
- Localization of the (TTAGGG)(n) telomeric repeat in the chromosomes of the pufferfish Tetraodon fluviatilis (Hamilton Buchanan) (Osteichthyes)
[Articolo su rivista]
Mandrioli, Mauro; Cuoghi, Barbara; Marini, Milena; Manicardi, Gian Carlo
abstract
In order to characterise the telomeric repeats of the pufferfish Tetraodon fluviatilis, fluorescent in situ hybridization (FISH) was carried out on metaphase chromosomes using a PCR generated probe (TTAGGG)(n). Distinct signals have been observed on the telomeres of all the chromosomes and hybridization signals appears of uniform size and intensity. Moreover FISH experiment does not evidence any interstitial non-telomeric signal. As non-telomeric FISH signal has been used as a marker for karyotype evolution and for determining the evolutionary status of fish species, it could be supposed that the absence of non telomeric signals makes T. fluviatilis (Tetraodontiformes) an evolutionary ancient species in respect to Perciformes.
1999
- NORs heteromorphism within a parthenogenetic lineage of the aphid Megoura viciae
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo; D., Bizzaro; U., Bianchi
abstract
In parthenogenetic females of a clone of the aphid Megoura viciae (Homoptera, Aphididae), more than 50% of the cells show heteromorphism between homologous NORs which are located on one telomeric region of the two X chromosomes. Using different techniques, such as staining with the CG-specific fluorochrome chromomycin A(3), silver staining and in-situ hybridization with an rDNA probe, we have shown that the observed heteromorphism is due to an unequal distribution of ribosomal genes between homologous NOR regions. The total number of rDNA genes per individual aphid remained constant. Moreover, the analysis of cells from single embroys has shown that the observed heteromorphism is not only intraclonal but also intraindividual. These data, together with the finding of X chromosomes connected by chromatin bridges between their NORs, allow us to suggest that mitotic unequal crossing over could be the main cause of NOR heteomorphism in this taxon.
1999
- Origin of DNA damage in ejaculated human spermatozoa
[Articolo su rivista]
Sakkas, D.; Mariethoz, E.; Manicardi, Gian Carlo; Bizzaro, D.; Bianchi, P. G.; Bianchi, U.
abstract
The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown, Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. Ln this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.
1999
- The role of rDNA genes in X chromosome association in the aphid Acyrthosipon pisum.
[Articolo su rivista]
Mandrioli, Mauro; Manicardi, Gian Carlo; D., Bizzarro; M., Giusti; U., Bianchi
abstract
Silver staining of mitotic metaphases of the aphid A. pisum reveals the presence of argentophilic bridges connecting the two X chromosomes. The presence of nucleolar material connecting sex chromosomes seems to be quite a common phenomenon in organisms belonging to very different phyla, and suggests a role of nucleolar proteins in chromosome association and disjunction. In somatic cells of A. pisum, bridges connecting X chromosomes are detectable not only after silver staining but also after CMA3 staining. This finding suggests that GC rich DNA is involved in this type of association. Molecular analysis of rDNA intergenic spacers shows several 247 bp repeats containing short sequences having a high level of homology with the chi sequence of Escherichia coli and with the consensus core region of human hypervariable minisatellites. Moreover, each 247 bp repeat presents a perfect copy of a promoter sequence for polymerase I. These aphid repeats show structural homologies with a 240 bp repeat, which is considered to be responsible for sex chromosome pairing in Drosophila, not only in view of their common presence within rDNA spacers but also for their length and structure. The presence of chi sequences in the IGS of A. pisum, by promoting unequal crossing-over between rDNA genes, could thus give rise to the nucleolar organizing region (NOR) heteromorphism described in different aphid species. Although X pairing at NORs is fundamental in aphid male determination, the presence of heteromorphism of rDNA genes does not inhibit male determination in the A. pisum clone utilized for our experiments.
1998
- Cytogenetic analysis on the gall generation of two aphid species: Tetraneura nigriabdominalis and T-ulmi
[Articolo su rivista]
Galli, Ermanno; Manicardi, Gian Carlo
abstract
We carried out a comparative cytogenetic study on gall-forming generations of Tetraneura nigriabdominalis and Tetraneura ulmi using NOR and C-banding followed by Giemsa, Chromomycin A(3) (CMA(3)) and DAPI staining reactions in order to improve our knowledge of these species and better define their identification. In both species, C-banding revealed that heterochromatin was not equilocated on each chromosome. Moreover, in T. nigriabdominalis the C-banding technique identified B chromosomes. The different response to the CMA(3) and DAPI staining reactions after C-banding points out heterogeneity of the heterochromatic DNA composition in both species. Moreover, both CMA(3) and silver staining revealed heteromorphism of NORs.
1998
- DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays.
[Articolo su rivista]
Manicardi, Gian Carlo; Tombacco, A.; Bizzaro, D.; Bianchi, U.; Bianchi, P. G.; Sakkas, D.
abstract
The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A(3) positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A(3)
1998
- In-situ competition between protamine and fluorochromes for sperm DNA
[Articolo su rivista]
D, Bizzaro; Manicardi, G.; G, BIANCHI P.; U, Bianchi; E, Mariethoz; D, Sakkas
abstract
In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A(3) (CMA(3)) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA, or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA(3) or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA(3) fluorescence, while the intensity of DAPI staining was decreased to similar to 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA(3) fluorescence is a strong indicator of the protamination state of spermatozoa.
1998
- Molecular and cytogenetic characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae
[Abstract in Rivista]
Bizzaro, D; Mandrioli, Mauro; Manicardi, Gian Carlo; Gionghi, D; Bassoli, L; Bianchi, U.
abstract
The paper describes chromosomal localization of a a highly repeated DNA sequence in the peach potato aphid Myzus persicae.
1998
- Occurrence of NORs heteromorphism in the two X chromosomes of different aphid species
[Capitolo/Saggio]
Manicardi, Gian Carlo; Mandrioli, Mauro; D., Bizzaro; U., Bianchi
abstract
In this paper we review recent work concerning both cytogenetic and molecular features of constitutive heterochromatin in the holocentric chromosomes of different aphid species. Sections include an extensive survey regarding chromosomal localization and DNA base composition of C-positive areas together with a cytological and molecular analysis of nucleolar organizer regions. The possible role of mitotic crossing over on genetic variability in parthenogenetic lineages is also briefly reviewed.
1998
- Occurrence of NORs heteromorphism of the two X chromosomes in different aphid species.
[Relazione in Atti di Convegno]
Manicardi, Gian Carlo; Bizzaro, D; Mandrioli, Mauro; Bianchi, U; Crema, R.
abstract
The chromosomal localization of the rDNA has been determined in embryo cells of four aphid species (Megoura viciae, Acyrthosiphon pisum, Myzus persicae, Aphis sambuci) by staining with Chromomycin A3, a fluorochrome specific for GC-rich DNA, by silver staining and by in situ hybridization with a probe obtained by PCR amplification of 28S rDNA obtained from A. pisum. All these techniques showed that in all the examined species the ribosomal cistrons are located in one telomeric position on the X chromosomes. This localization of NORs seems to be a general phenomenon in aphids whereas autosomal NORs have been reported for Schoutedenia ralumensis only. Our data show a certain amount of heteromorphism between homologous NORs. This condition, already reported for some aphid species, is an unusual phenomenon in other animal species. The origin of this rDNA heteromorphism is still unclear. It may be caused by differential gene amplification, unequal crossing-over, unequal sister chromatid exchange or unequal mitotic crossing-over. The last hypothesis may be supported by the frequent observation of X connected by NORs bearing telomeres in mitotic metaphases.
1998
- Patterns of DNase I sensitivity in the holocentric chromosomes of the aphid Megoura viciae
[Articolo su rivista]
Manicardi, Gian Carlo; Mandrioli, Mauro; D., Bizzaro; Bianchi, Umberto
abstract
Using the in situ nick translation technique, we looked for the presence of DNase I sensitive sites in Megoura viciae chromosomes, to study the distribution of active or potentially active genes in aphids, a group of insects possessing holocentric chromosomes. Cytological preparations obtained by the spreading of embryo cells were treated in situ with increasing concentrations (ranging from 5 to 200 ng/mL) of DNase I. At DNase I concentrations below 50 ng/mL, only one hypersensitive site was observed, and this was located on a telomeric region of the X chromosome that contains transcriptionally active nucleolar organizing regions, as assayed by silver staining. Interestingly, at intermediate concentrations of DNase. the incorporation of biotinylated nucleotide occurred uniformly throughout all chromosomes, whereas at concentrations above 100 ng/mL, a C-like banding pattern was produced. Our data differ from results obtained with mammalian, frog, and grasshopper chromosomes, where it was found that DNase I nicking is concentrated at the distal regions of all chromosomes.
1998
- Sex chromosome pairing: the role of rDNA genes in the aphid Acyrthosiphon pisum
[Abstract in Rivista]
Mandrioli, Mauro; Bizzaro, D; Manicardi, Gian Carlo; Bianchi, U.
abstract
The paper describes the role of rDNA genes in sex chromosome pairing in the aphid Acyrthosiphon pisum.
1998
- Silver staining as a new banding technique to identify aphid chromosomes
[Articolo su rivista]
Manicardi, Gian Carlo; D., Bizzaro; Mandrioli, Mauro; U., Bianchi
abstract
The karyotype of the aphid Aphis sambuci has been studied by means of C-banding, fluorochrome staining and AgNO3 staining. Heterochromatic areas are confined exclusively to the X chromosomes. Interestingly, silver staining produces a clear-cut longitudinal differentiation of all chromosomes, which allows reliable identification of homologues.
1998
- Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development
[Articolo su rivista]
Sakkas, D.; Urner, F.; Bizzaro, D.; Bianchi, P. G.; Manicardi, Gian Carlo; Shoukir, Y.; Campana, A.
abstract
This paper illustrates the effect on fertilization and embryo development of sperm nuclear DNA damage and altered chromatin structure.
1997
- Chromatin packaging anomalies and DNA damage in human sperm: their possible implications in the treatment of male factor infertility.
[Relazione in Atti di Convegno]
Sakkas, D.; Bianchi, P. G.; Manicardi, Gian Carlo; Bizzaro, D.; Bianchi, U.
abstract
The paper illustrates the relationships between chromatin packaging anomalies and DNA damage in human sperm and their possible implications in the treatment of male factor infertility.
1996
- Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa.
[Articolo su rivista]
Bianchi, P. G.; Manicardi, Gian Carlo; Urner, F.; Campana, A.; Sakkas, D.
abstract
This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, > 75% of the macrocephalic sperm fluoresced in all patients. In contrast, a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (> 70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozoa.
1996
- Chromosomal localization of a highly repeated EcoRI DNA fragment in Megoura viciae (Homoptera, Aphididae) by nick translation and fluorescence in situ hybridization
[Articolo su rivista]
D., Bizzaro; Manicardi, Gian Carlo; U., Bianchi
abstract
To investigate the genome of the aphid Megoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases. EcoRI digestion produced a highly repeated DNA fragment, about 600 bp long. The contribution of this EcoRI element to the total genome of M. viciae was estimated at about 6% by means of densitometric scanning of agarose gel photographs. The chromosomal localization of this fragment, investigated by fluorescent in situ hybridization (FISH), constantly showed one large and two narrower fluorescent bands located on the X chromosome, all corresponding to C-positive heterochromatic areas. These results are in full accordance with the data obtained by in situ nick translation experiments carried out after EcoRI digestion, and clearly demonstrate that a substantial amount of M. viciae heterochromatin consists of EcoRI fragments which are mainly located on the X chromosome. Using the EcoRI restriction fragment as a molecular probe may prove to be a practical tool for the investigation of taxonomic and evolutionary relationships in this group of insects.
1996
- Heterochromatin heterogeneity in the holocentric X chromatin of Megoura viciae (Homoptera, Aphididae)
[Articolo su rivista]
MANICARDI, Gian Carlo; D., Bizzaro; GALLI, Ermanno; U., Bianchi
abstract
Holocentric chromosomes, prepared by spreading embryo cells obtained from Megoura viciae parthenogenetic females, have been C-banded, enzymatically digested in situ using the specific endonucleases DdeI (C down arrow TNAG), DraI (TTT down arrow AAA), Tru9I (TT down arrow AA), and CfoI (GCG down arrow C), and subsequently stained with Giemsa, DAPI, CMA(3), and AgNO3. We observed that the X chromosome had the best defined banding patterns. In the M. viciae X chromosome there is a certain amount of heterogeneity in heterochromatic DNA composition. In fact, the CC-rich NOR-associated heterochromatin differs from other heterochromatic bands that are characterized by AT-rich DNAs. Our data also indicate that, in M. viciae holocentric chromosomes, all heterochromatic blocks are accessible to in situ enzyme attack, the only limit to the digestion being the presence or absence of recognition targets. This is an interesting point, since, in monocentric chromosomes, it is well known that in situ endonuclease digestion is heavily affected not only by DNA base composition but also by chromatin compactness that may limit enzyme accessibility to their specific targets.
1996
- Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection
[Articolo su rivista]
Sakkas, D; Urner, F; Bianchi, Giampaolo; Bizzaro, D; Wagner, I; Jaquenoud, N; Manicardi, Gian Carlo; Campana, A.
abstract
In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality, Sperm chromatin packaging quality was assessed using the chromomycin A(3) (CMA(3)) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of >20%, CMA(3) fluorescence of <30% and exhibit endogenous nicks in <10% of their spermatozoa, When patients were separated according to these values no difference was observed in their fertilization rates after ICSI, When the unfertilized ICSI oocytes were examined, we found that patients with CMA(3) fluorescence of <30% and nicks in <10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed, In contrast, patients with higher CMA(3) and nick values had a significantly higher number, 41.2 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. Sperm morphology did not show any such pattern, The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA(3) fluorescence or nicks, In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.
1996
- Use of the Guanine-Cytosine (GC) specific fluorochrome, Chromomycin A3, as an indicator of poor sperm morphology.
[Articolo su rivista]
Bianchi, P. G.; Manicardi, Gian Carlo; Bizzaro, D.; Campana, A.; Bianchi, U.; Sakkas, D.
abstract
Purpose: We have previously postulated that the chromomycin A(3) (CMA(3))fluorochrome allows an indirect visualization of sperm chromatin packaging quality and partially denatured sperm DNA, In this study we investigate the relationship between CMA(3) positivity and sperm morphology. We also present data on the association between sperm morphology and the presence of endogenous nicks in sperm DNA.Methods: Semen samples were examined from 81 males of the couples who were consulting for infertility treatment. CMA(3) fluorescence was assessed for all samples, while in 24 sperm samples we also examined for the presence of endogenous nicks in the sperm DNA,Results: When sperm morphology was less than 20% normal in a patient, the level of CMA(3) fluorescence and presence of endogenous nicks were significantly higher than in patients with a higher incidence of morphologically normal sperm.Conclusions: CMA(3) could be used as an adjunct to the assessment of morphology as an evaluation method for poor sperm, Its value in predicting fertilizing ability when using either SUZI or ICSI awaits to be answered.
1995
- Approccio citogenetico e molecolare allo studio della cromatina in cromosomi olocentrici-olocinetici.
[Capitolo/Saggio]
Bianchi, U.; Manicardi, Gian Carlo; Bizzaro, D.
abstract
Il lavoro illustra i risultati ottenuti sviluppando un approccio citogenetico e molecolare allo studio della cromatina in cromosomi olocentrici-olocinetici negli afidi.
1995
- Cytofluorimetric evidence for differential genome endoreplication in the cluster neurons of Lophius piscatorius L. (Osteichthyes, Lopiiformes)
[Articolo su rivista]
D., Sassi; Manicardi, Gian Carlo; Mola, Lucrezia; I., Benedetti
abstract
Quantitative microfluorometric evaluation indicated that, in Lophius piscatorius, the DNA content of large neurons of a cluster located at the boundary between the medulla oblongata and the spinal cord varied from 4C to more than 5000C. This finding must be considered exceptional for the nervous system of Vertebrates. DNA contents were correlated to both nuclear and animal size. Utilization of AT and GC specific fluorochromes showed that the increase in DNA content is due to differential genome amplification involving GC-rich DNA sequences.
1995
- DNA content in the nurse cell nuclei of viviparous and oviparous females of Megoura viciae
[Articolo su rivista]
Manicardi, Gian Carlo; Galli, E.; Malavasi, A.; BONVICINI PAGLIAI, A. M.
abstract
Microfluorometric evaluations of DNA content carried out in both viviparous and oviparous females of Megoura viciae have shown that the increased nuclear volume of the nurse cell nuclei is due to repeated duplications of the whole genome. DNA overreplication begins, together with the first appearance of the ommatidia, 3 days before birth both in viviparous and oviparous female embryos, but it proceeds at different rates in the different reproductive categories. In oviparous females, 11 replication cycles take place (maximum DNA content 2048C), whereas in viviparous females only a few DNA replications occur (maximum DNA content 16C). The relationship we found between the increased DNA content in nurse cell nuclei and development of ommatidia suggests that the switch mechanism determining the beginning of DNA overreplication may be directly influenced by photoperiod effects on the developing embryos.
1995
- NUCLEAR-DNA CONTENT IN GASTROTRICHA
[Articolo su rivista]
Balsamo, M; Manicardi, Gian Carlo
abstract
A cytofluorometric evaluation of nuclear DNA content was carried out on fifteen species of Gastrotricha. Genome size, ranging from 0.05 to 0.63 pg, appears uniform in this group and rather low compared to values found in other lower Metazoa. Differences in DNA content between the two orders of the phylum, Macrodasyida and Chaetonotida, which differ greatly in morphology and reproductive biology, are not evident. From these data, a polyploid condition of obligatory parthenogenetic Chaetonotida seems unlikely.
1995
- Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationships to Chromomycin A3 accessibility.
[Articolo su rivista]
Manicardi, Gian Carlo; Bianchi, P. G.; Pantano, S.; Azzoni, P.; Bizzaro, D.; Bianchi, U.; Sakkas, D.
abstract
During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A(3) (CMA(3)) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA(3) and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA(3) stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa.Human spermatozoa from 25 different samples showed values of sensitivity to the CMA(3) fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA(3) fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA(3) showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.
1995
- Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa.
[Articolo su rivista]
Sakkas, D.; Manicardi, Gian Carlo; Bianchi, P. G.; Bizzaro, D.; Bianchi, U.
abstract
Mammalian spermiogenesis involves the replacement of histones by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A(3) (CMA(3)) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA(3) fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96%) to CMA(3), decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA(3) accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA(3)- positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA(3) fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm.We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.
1994
- CYTOGENETIC INVESTIGATIONS ON THE HOLOCENTRIC CHROMOSOMES OF TETRANEURELLA-AKINIRE (SASAKI) (HOMOPTERA, PEMPHIGIDAE)
[Articolo su rivista]
Manicardi, Gian Carlo; Gautam, Dc
abstract
The karyotype of the root aphid Tetraneurella akinire was analysed by means of C, G, and NOR banding. The overall base pair distribution was investigated after CMA, and DAPI fluorochrome staining. Both C and G treatments revealed a well defined banding pattern on the X and A1 chromosomes. C-bands showed a marked polymorphism in both size and number, even within the same individual. NOR banding produced a precipitation of silver on one telomere of the X chromosome, which also showed bright fluorescence after CMA, staining. Treatment with DAPI uniformly stained all chromosomes. Neither CMA3 nor DAPI showed bright fluorescence overlapping the C-positive bands. It can be concluded that T. akinire chromosomes present a cluster of GC-rich DNA sequences located on one telomere of the X chromosome, whereas the AT - sequences are regularly arranged along the chromosome axes and not grouped into well defined clusters.
1994
- Cytological and electrophoretic analysis of DNA methylation in the holocentric chromosomes of Megoura viciae (Homoptera, Aphididae).
[Articolo su rivista]
MANICARDI, Gian Carlo; BIZZARO, D.; AZZONI, P.; BIANCHI, U.
abstract
Chromosomal and purified DNA methylation patterns were determined in the holocentric chromosomes of Megoura viciae by treatment with MspI and HpaII. Both enzymes produced a clear C-like banding pattern but widely digested one telomere of the X chromosome, which appeared as heterochromatic after C-banding treatment and brightly fluorescent after chromomycin A(3) staining. Quantitative microfluorometric evaluations of DNA extraction performed on cytological preparations showed that both isoschizomers resulted in the same DNA extraction (about 30%). Contrary to what was found by in situ endonuclease treatment, the electrophoretic patterns of purified and digested DNA showed that digestion with MspI was slightly more extensive than that with HpaII in a zone of fragments ranging from 23 to 9 kb. This result indicates that aphid chromatin is not wholly unmethylated. The discrepancy between electrophoretic and cytological data has been explained by taking into consideration that DNA fragments with high molecular weights could be cleaved in situ by the enzymes but not extracted from the chromatin.
1994
- Faunistic study in the karst complex of Frasassi (Genga, Italy)
[Articolo su rivista]
Bertolani, Roberto; Manicardi, Gian Carlo; Rebecchi, Lorena
abstract
La fauna cavernicola del complesso carsico di Frasassi è stata studiata per avere infromazioni dettagliate sulla popolazione aniamle ipogea.
1994
- Faunistic study in the karst complex of Frasassi.
[Articolo su rivista]
Bertolani, Roberto; Manicardi, Gian Carlo; Rebecchi, L.
abstract
Data regarding faunistic analyses in the karst complex of Frasassi are reported.
1994
- New data on the nuclear DNA content in some species of tardigrades.
[Capitolo/Saggio]
Bertolani, Roberto; S., Garagna; Manicardi, Gian Carlo; Rebecchi, Lorena; C. A., Redi
abstract
In the chapter the Feulgen DNA content was evlauted in several species of tardigrades. A small genome size and poliploidy was confirmed.
1993
- A cytochemical study of mature mouse spermatozoa after C-banding treatment.
[Articolo su rivista]
Bianchi, P. G.; Manicardi, Gian Carlo; Bizzaro, D.; Sakkas, D.; Bianchi, U.
abstract
Heterochromatin in mature mouse spermatozoa has been investigated by using C-banding treatment followed by either i) Giemsa staining, ii) staining with DAPI, a fluorochrome specific for AT rich DNA and iii) by using quantitative DNA measurements [Acriflavine-Feulgen, DAPI and Ethidium Bromide (EB)]. We have shown that a fraction of the mature sperm chromatin is affected by C-banding treatment. The sperm chromatin treated with Ba(OH)2 and stained with EB doubled fluorescence emission values found in untreated control preparations. This experimental result clearly shows that the use of intercalating fluorochromes, such as EB, is unsuitable for quantitative evaluation of highly condensed DNAs.
1993
- Cytofluorimetric determination of DNA content in large neurons of Lophius piscatorius L. (Osteichthyes, Lopiiformes)
[Articolo su rivista]
I., BENEDETTI; MANICARDI, Gian Carlo; MOLA, Lucrezia
abstract
The central nervous system of Lophius piscatorius has a cluster of large neurons located at the boundary between the medulla oblongata and spinal cord. The DNA content of these neurons was evaluated by microfluorimetric methods. Results demonstrated a DNA content ranging from a minimum of 8C in the smaller to over 1000C in the larger neurons of L. piscatorius cluster. These data are exceptional for the nervous system of Vertebrates, and known only for the giant neurons of Mollusca.
1993
- EFFECT OF DEOXYRIBONUCLEIC-ACID PROTAMINATION ON FLUOROCHROME STAINING AND IN-SITU NICK-TRANSLATION OF MURINE AND HUMAN MATURE SPERMATOZOA
[Articolo su rivista]
Bianchi, Pg; Manicardi, Gian Carlo; Bizzaro, D; Bianchi, U; Sakkas, D.
abstract
A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) Can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.
1993
- Indagine sul DNA contenuto nel nucleo dei neuroni del cluster di Lophius piscatorius L. (Lophiiformes, Osteichthyes).
[Poster]
D., Sassi; Mola, Lucrezia; Manicardi, Gian Carlo; I., Benedetti
abstract
Sono state condotte indagini morfometriche e citofluorimebntriche sui neuroni sopramidollari riuniti in cluster di l. piscatorius. Il contenuto in DNA è sempre maggiore di 2C eraggiunge valori mai riscontrati prima in neuroni di vertebrati.
1993
- KARYOTYPE ANALYSIS AND DNA CONTENT IN 2 SPECIES OF ROOT APHIDS
[Articolo su rivista]
Gautam, Dc; Bizzaro, D; Manicardi, Gian Carlo
abstract
In this paper we describe the karyotypes of two aphid species, Tetraneurella akinire and Anoecia nemoralis, found on the roots of Graminacee. Karyotype analysis in T. akinire have shown variations in chromosome number which are discussed in relation to the evolution of aphid karyotypes. Moreover, we estimated the DNA content in somatic cells of both species. Since emission values observed in mitotic plates of T. akinire with 14-15-16 chromosomes were completely overlapping, we can conclude that these karyotype variations are solely due to chromosome fragmentation.
1992
- Amplification of GC-rich DNAs in neuronal nuclei of Planorbarius corneus (L.) (Mollusca, Pulmonata).
[Articolo su rivista]
MANICARDI, Gian Carlo; BIZZARO, D.; SONETTI, D.; LOMBARDO, F.; BIANCHI, U.
abstract
Quantitative microfluorometric evaluation of DNA content in nerve cells of the Pulmonate Gastropod Planorbarius corneus has indicated that the increase in nuclear volume is due to DNA amplification. Indeed, it has been observed that the DNA contents are scattered at random between 2C and 1,000C values. This is not in agreement with the occurrence of repeated duplications of the whole genome. Furthermore, chromatin photo-oxidation, a technique useful in discriminating GC-rich from AT-rich DNAs, suggests that DNA amplification involves GC-rich sequences.
1992
- New approaches to the study of aphids chromosomes.
[Relazione in Atti di Convegno]
Manicardi, Gian Carlo; Bizzaro, D.; Aloisi, P.; BONVICINI PAGLIAI, A. M.; Bianchi, U.
abstract
The paper describes the advantages in using new approaches to the study of aphids holocentric chromosomes.
1992
- THE NUCLEAR-DNA CONTENT OF LARGE NEURONS OF LOPHIUS-PISCATORIUS L (OSTEICHTYES)
[Abstract in Rivista]
Benedetti, I; Manicardi, Gian Carlo; Mola, Lucrezia
abstract
.
1992
- The nuclear DNA content of large neurons of Lophius piscatorius L. (Osteichthyes).
[Poster]
I., Benedetti; Manicardi, Gian Carlo; Mola, Lucrezia
abstract
The DNA content of supramedullary neurons of L. piscatorius, measured on slides treated with Ethidium bromide and acriflavine Feulgen, ranged from 8C in the smaller neurons to 1024 C in the larger neurons.
1991
- Characterization of Acyrthosiphon pisum Harris (Homoptera, Aphididae) karyotype using G and C banding.
[Relazione in Atti di Convegno]
Manicardi, Gian Carlo; Bizzaro, D.; BONVICINI PAGLIAI, A. M.; Bianchi, U.
abstract
The paper deals with the characterization of Acyrthosiphon pisum Harris (Homoptera, Aphididae) karyotype using G and C banding.
1991
- Chromosome banding in Aphids: G, C, AluI and HaeIII banding patterns in Megoura viciae (Homoptera, Aphididae).
[Articolo su rivista]
Manicardi, Gian Carlo; Gautam, D. C.; Bizzaro, D.; Guicciardi, E.; BONVICINI PAGLIAI, A. M.; Bianchi, U.
abstract
The holocentric mitotic chromosomes of Megoura viciae, a species that has been little studied cytogenetically to date, have been characterized by applying G, C, AluI, and HaeIII banding techniques. C bands have shown the best defined patterns, particularly on the X chromosome. This chromosome, on the other hand, behaved as the most reactive to the various treatments. Uncondensed, prometaphase X chromosomes showed a number of heterochromatic bands, interspersed among the euchromatin, which fused together during metaphase condensation. AluI and HaeIII treatments also produced reproducible banding patterns. These data permit an accurate identification of the X chromosome as well as of the autosomal pairs 1 and 2, and facilitate the construction of nonambiguous karyotypes. They will also stimulate studies on the organization of chromatin in holocentric, holokinetic chromosomes. Finally they could also promote research on chromosomal rearrangements that have occurred during the course of speciation and evolution of aphids, since these kinds of events may be significantly affected by the condition of chromosomal holocentrism.
1989
- Cenni sul popolamento animale di alcune grotte del Complesso Carsico di S. Ninfa (Tp)
[Capitolo/Saggio]
Bertolani, Roberto; Manicardi, Gian Carlo
abstract
L'articolo descrive i dati relativi ad una campagna di raccolta di materiale biologico condotta in alcune grotte nei gessi dell'area carsica di S. Ninfa.
1989
- Two new species of Eutardigrades (Tardigarda) from Canada with consideration on environmental morphological relationship in soil dwelling tardigrades.
[Articolo su rivista]
Manicardi, Gian Carlo
abstract
The paper deals with the description of two new species of Eutardigrades (Tardigarda) from Canada with consideration on environmental morphological relationship in soil dwelling tardigrades.
1988
- Effetti delle pratiche agronomiche sui microartropodi del terreno del suolo: l'aratura
[Poster]
Mola, Lucrezia; Manicardi, Gian Carlo; R., Lumetti; Sabatini, Maria Agnese; Bertolani, Roberto; P., Loro
abstract
sono stati messi a confronto due campi contigui arati in profondità per diversi anni; in uno di essi l'aratura profonda è stata sostituita con un 'aratura superficiel (10-15 cm). A 4 anni dalla sostituzione il numero sia degli acari che dei collemboli è significativamente maggiore nel campo arato solo superficialmente.
1987
- Determination of ATP and ATPase activity in Macrobiotus richtersi and in Pseudobiotus megalonix (Tardigrada)
[Capitolo/Saggio]
Davolio, E.; Manicardi, Gian Carlo; Volpi, Nicola
abstract
Determination of ATP and ATPase activity in Macrobiotus richtersi and in Pseudobiotus megalonix (Tardigrada)
1987
- First contribution to the knowledge of alpine grassland tardigrades.
[Capitolo/Saggio]
Manicardi, Gian Carlo; Bertolani, Roberto
abstract
The paper describes the composition of tardigrades fauna in alpine grasslands.
1987
- Macrobiotus pseudohufelandi Iharos as a model for cytotaxonomic study in populations of eutardigrades (Tardigrada).
[Articolo su rivista]
Bertolani, R.; Garagna, S.; Manicardi, Gian Carlo; Redi, C. A.
abstract
The paper illustrate a cytophotometric approach able to furnish cytotaxonomic information regarding Macrobiotus pseudohufelandi Iharos (Tardigrada).
1987
- Tardigradi della Riserva Naturale del Torricchio e dei Monti Sibillini.
[Capitolo/Saggio]
Bertolani, Roberto; Manicardi, Gian Carlo; Gibertoni, D.
abstract
L'articolo presenta i risultati di una ricerca faunistica relativa allo studio della Tardigradofauna della Riserva Naturale del Torricchio e dei Monti Sibillini.
1986
- New cases of hermaphroditism in tardigrades.
[Articolo su rivista]
Bertolani, Roberto; Manicardi, Gian Carlo
abstract
The paper describes evidences of hermaphdroditic reproduction in some tardigrades.
1986
- Notulae Oribatologicae XXXVIII. On the first european record of an Eohypochthonius species in Italy (Acarida, Oribatida).
[Articolo su rivista]
Bernini, F.; Manicardi, Gian Carlo; Avanzati, A. M.
abstract
The paper illustrates the first european record of an Eohypochthonius species in Italy (Acarida, Oribatida).
1985
- Determination of ATP in Macrobiotus richtersi using luminometric techniques
[Poster]
Davolio, E.; Manicardi, Gian Carlo; Volpi, Nicola
abstract
Determination of ATP in Macrobiotus richtersi using luminometric techniques