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Stefano CASSANELLI
Ricercatore Universitario
Dipartimento di Scienze della Vita sede ex-Agraria
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Pubblicazioni
2023
- A viability qPCR protocol to assess the efficacy of a heat
treatment to sanitize carrot seeds from Candidatus
Liberibacter solanacearum
[Articolo su rivista]
Ben Othmen, Soukaina; Conti Nibali, Gianmarco; Cassanelli, Stefano; Giovanardi, Davide; Stefani, Emilio
abstract
Seeds may harbour plant pathogens and,
therefore, may be regarded as a possible pathway for
their dissemination along trade routes worldwide.
Heat treatment appears to be a proper seed sanitation
method to comply with the requirement of phytosanitary
measures. Carrot seeds have been supposed as a
pathway for the dissemination of Candidatus Liberibacter
solanacearum, although vertical pathogen
transmission is still under debate: nonetheless, such
seeds may be conveniently heat treated as a sanitation
method. We used a dry thermal treatment (50 ± 1 °C
for 72 h) to sanitize seeds from Ca. Liberibacter solanacearum
and, since this bacterium is not cultivable,
we additionally implemented and evaluated a viability
qPCR protocol able to detect only viable cells,
therefore confirming seed sanitation. The population
of the pathogen was estimated through a plasmid
calibration curve. The infection of Ca. Liberibacter
solanacearum was quantified in two positive samples
at approx. 5 × 106
cells gram −1 of seed. Seed sample
tested with monoazides (EMA and PMA) qPCR clearly indicated that seed-associated bacteria lost
their viability during the heat treatment, thus confirming
seed sanitation. Finally, such heat treatment did
not affect seed viability and seed quality: conversely,
a remarkable reduction of seed saprophytes was
observed, together with a better vigour germination
performance.
2022
- Better under stress: Improving bacterial cellulose production by Komagataeibacter xylinus K2G30 (UMCC 2756) using adaptive laboratory evolution
[Articolo su rivista]
Anguluri, K.; La China, S.; Brugnoli, M.; Cassanelli, S.; Gullo, M.
abstract
Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1-25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production.
2022
- Candidate Acetic Acid Bacteria Strains for Levan Production
[Articolo su rivista]
Anguluri, Kavitha; LA CHINA, Salvatore; Brugnoli, Marcello; DE VERO, Luciana; Pulvirenti, Andrea; Cassanelli, Stefano; Gullo, Maria
abstract
In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.
2021
- Evaluation of insecticide efficacy and insecticide adaptive response in italian populations of drosophila suzukii
[Articolo su rivista]
Civolani, S.; Vaccari, G.; Caruso, S.; Finetti, L.; Bernacchia, G.; Chicca, M.; Cassanelli, S.
abstract
Monitoring sensitivity to insecticides is crucial to prevent outbreaks of invasive pests characterized by high reproductive and adaptive potential such as the Drosophila suzukii (Matsumura) (Diptera Drosophilidae). The aim of the present study was to investigate the possible appearance of resistance to cyantraniliprole, deltamethrin and spinosad. Field trials on commercial sweet cherry orchards in Northern Italy showed that two out of six strains were not fully controlled using cyantraniliprole and deltamethrin, while spinosad was thoroughly effective. At the bioassay, two populations showed a decrease in deltamethrin and cyantraniliprole susceptibility (LC50 values 12.7-21.0 and 3.4-5.8 times higher than those from the untreated populations, respectively). Biochemical analyses revealed that low resistance to the pesticides was associated with high monooxygenase and carboxylesterase activities (range 2.68-4.37-and 1.97-2.73 times higher than in the wild population). A dose-dependent increase in cytochrome P450 monoox-ygenase Cyp12d1 and ryanodine receptor gene expression was found when a strain with low resistance to cyantraniliprole in field trials was treated with increasing dosages of the diamide in bioassays. No mutations were detected in voltage-gated sodium channel and ryanodine receptor genes, which accounted for the reduction in pyrethroid and diamide susceptibility in other pests. After 8 generations of selection, starting from a susceptible population, the LC50 values of cyantraniliprole and deltamethrin were increased 2.2 and 25.0 fold, respectively, compared with the unselected colony. In contrast, no selection was possible for spinosad. Our study would suggest that spotted wing drosophila, upon continued selective pressure, are more prone to develop low resistance to cyan-traniliprole and deltamethrin than spinosad. The adaptive response relies on detoxifying activities of monooxygenases and increased Cyp12d1 and ryanodine receptor gene expression.
2021
- Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts
[Articolo su rivista]
Solieri, L; CassanelliS, ; Huff, F; Barroso, L; Branduardi, P; Louis, Ej; Morrissey, Jp
abstract
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
- Modulation of Drosophila suzukii type 1 tyramine receptor (DsTAR1) by monoterpenes: a potential new target for next generation biopesticides
[Articolo su rivista]
Finetti, L.; Ferrari, F.; Calo, G.; Cassanelli, S.; De Bastiani, M.; Civolani, S.; Bernacchia, G.
abstract
This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes. A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 μM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80). The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators. Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1. This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes. Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.
2019
- A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii
[Articolo su rivista]
Bizzarri, Melissa; Cassanelli, Stefano; Dušková, Michala; Sychrová, Hana; Solieri, Lisa
abstract
The so‐called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase‐mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418‐resistant mutants previously constructed by replacing the MATαP expression locus with the loxP–kanMX–loxP cassette.
2019
- Interplay of Chimeric Mating-Type Loci Impairs Fertility Rescue and Accounts for Intra-Strain Variability in Zygosaccharomyces rouxii Interspecies Hybrid ATCC42981
[Articolo su rivista]
Bizzarri, Melissa; Cassanelli, Stefano; Bartolini, Laura; Pryszcz, Leszek P.; Dušková, Michala; Sychrová, Hana; Solieri, Lisa
abstract
The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii. Recently, different mating-type-like (MTL) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MATa/MATα expression loci, in contrast to MATα in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MATα expressed locus by gene deletion. The resulting MATa/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HMLα cassette masks the loss of heterozygosity at the MAT locus. We also found that MATα deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast.
2019
- Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections
[Articolo su rivista]
De Vero, Luciana; Boniotti, Maria Beatrice; Budroni, Marilena; Buzzini, Pietro; Cassanelli, Stefano; Comunian, Roberta; Gullo, Maria; Logrieco, Antonio F.; Mannazzu, Ilaria; Musumeci, Rosario; Perugini, Iolanda; Perrone, Giancarlo; Pulvirenti, Andrea; Romano, Paolo; Turchetti, Benedetta; Varese, Giovanna Cristina
abstract
Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs.
2018
- Cloning, molecular characterization and tissue exPression of an octoPamine/tyramine recePtor from sPotted wing drosoPhila (DROSOPHILA SUZUKII)
[Abstract in Atti di Convegno]
Finetti, Luca; Bernacchia, Giovanni; Cassanelli, Stefano; Civolani, Stefano
abstract
Spotted wing Drosophila (Drosophila suzukii) is a polyphagous pest arrived in Europe in 2009 able to infest a growing number of fruit and vine
species, causing considerable economic damage. D. suzukii grows very rapidly (seven to fifteen generations per year) and shows a remarkable
ability to adapt to climatic conditions and to new host plants. These characteristics make its populations particularly difficult to control. Octopamine
(OA) and tyramine (TA) biogenic amines are present in traces in vertebrates, while in invertebrates they act as substitutes for adrenaline and
noradrenaline. Indeed, these amines regulate numerous physiological processes in insects. They exert their effects by binding to specific receptor
proteins that belong to the superfamily of G-protein coupled receptors (GPCRs). In this work, we have isolated complementary DNA (cDNA) coding
for an amine receptor from Drosophila suzukii (DsTyr). The cloned cDNA is about 1.8kb long and encodes for a 601 amino acids protein. This
polypeptide presents the classical seven transmembrane domains as revealed by hydropathic profile analysis. BLAST analysis of the sequence
shows a high identity (>98%) to the octopamine/tyramine receptor from Drosophila melanogaster. DsTyr1 deduced sequence will be compared to
the amino acid sequence of octopamine/tyramine receptors from other insects. Furthermore, the various receptor sequences will be characterized
by phylogenetic analysis. The expression level of the receptor will be studied by qRT-PCR analysis in different parts of D. suzukii male and female
body (head, thorax and abdomen). With this work, we present a first structural and functional description of an octopamine/tyramine receptor from
Drosophila suzukii.
2018
- Corrigendum: Evidence of a female-produced sex pheromone in the european pear psylla, cacopsylla pyri (Bulletin of Insectology 71, 1, 57-64, 2018)
[Articolo su rivista]
Ganassi, S.; Germinara, G. S.; Pati, S.; Civolani, S.; Cassanelli, S.; Sabatini, M. A.; De Cristofaro, A.
abstract
Change “11,13-dimethylheptacosane” to “11,15-dimethylheptacosane” along all the text. Page 61, add figure 4, the experimental mass spectrum relevant to the peak 2 and the fragmentation scheme for the compound 11,15-dimethylheptacosane, similar to the one reported by Kenig et al. (1995). (Figure presented)Page 61, table 1, add this information in the footnotes “Molecular ions in the brackets were not visible in the spectrum, but could be inferred by the diagnostic ions”. Page 62, delete “11,13-dimethylheptacosane was reported as a sex pheromone component of Cataglyphis species (Hymenoptera Formicidae) (Dahbi et al., 1996).” Page 63, References, delete “DAHBI A., LENOIR A., TINAUT A., TAGHIZADEH T., FRANCKE W., HEFETZ A., 1996.-Chemistry of the postpharyngeal gland secretion and its implication for the phylogeny of Iberian Cataglyphis species (Hymenoptera: Formicidae).-Chemoecology, 7: 163-171.” Page 63, References, add “KENIG F., SINNINGHE DAMSTÉ J. S., KOCK-VAN DALEN A. C., RIJPSTRA W. I. C., HUC A. Y., DE LEEUW J. W., 1995.-Occurrence and origin of mono-, di-, and trimethylalkanes in modern and Holocene cyanobacterial mats from Abu Dhabi, United Arab Emirates.-Geochimica et Cosmochimica Acta, 59: 2999-3015.” We are grateful to our colleagues Jocelyn Millar and David Horton to alert us about a possible mistake in our identification of 11,13-dimethylheptacosane.
2018
- Draft genome sequences of the highly halotolerant strain Zygosaccharomyces rouxii ATCC 42981 and the novel allodiploid strain Zygosaccharomyces sapae ATB301T obtained using the MinION platform
[Articolo su rivista]
Bizzarri, M; Cassanelli, S; Pryszcz, Lp; Gawor, J; Gromadka, R; Solieri, L.
abstract
Here, we report draft genome sequences of the halotolerant and allodiploid strains Zygosaccharomyces rouxii ATCC 42981 and Zygosaccharomyces sapae ABT301T. Illumina and Oxford Nanopore MinION sequencing revealed genome sizes of 20.9 and 24.7 Mb, respectively. This information will be useful for deciphering the genetics of hybrid adaptation to high salt and sugar concentrations in nonconventional yeasts.
2018
- Evidence of a female-produced sex pheromone in the european pear psylla, cacopsylla pyri
[Articolo su rivista]
Ganassi, S.; Germinara, G. S.; Pati, S.; Civolani, S.; Cassanelli, S.; Sabatini, M. A.; de Cristofaro, A.
abstract
Cacopsylla pyri (L.) (Hemiptera Psyllidae) is one of the most important pests of pear orchards in Europe that reduces the market value of pears. Summerform C. pyri males significantly preferred odours from living females or female cuticular extracts in the absence of visual stimuli in a Y-tube olfactometer. Conversely, males as well as females did not show any preference for odours from specimen of the same sex. Electroantennogram recordings showed that female cuticular extracts elicit dose-dependent responses in male antennae suggesting the presence of volatile compounds capable to stimulate the male peripheral olfactory system. Gas-chromatography coupled with mass spectrometry revealed marked quantitative differences between male and female cuticular extracts regarding 13-methylheptacosane, 11,13-dimethylheptacosane, 2-methylheptacosane, and 3-methylheptacosane. These compounds were found in larger amounts in female extracts which suggests their role in male attraction.
2018
- Mating behaviour and dual mode communication of Pear Psylla CACOPSYLLA PYRI
[Poster]
Ganassi, Sonia; Civolani, Stefano; Finetti, Luca; Cassanelli, Stefano; De Cristofaro, Antonio
abstract
Cacopsylla pyri (L.) (Hemiptera, Psyllidae) is one of the most important pests of European pear, and its management generally depends on the use
of chemical insecticides, but C. pyri outbreaks are sometime observed. Ecological control strategies should be desirable and the knowledge of mating behavior is crucial to develop new ones. A multi-approaches research aimed to acquire knowledges about C. pyri mate finding. Electroantennographic (EAG) analyses and olfactometric bioassays were used to evaluate the activity of intraspecific semiochemicals on C. pyri. The EAG amplitudes revealed that volatile compounds, present in female cuticular extracts, elicited dose-dependent responses in males, indicating that these compounds were able to stimulate the male olfactory system. In behavioral bioassays, living females and female cuticular extracts, attracted summerform males in a highly significant manner. Gas chromatography-mass spectrometry revealed that 13-methylheptacosane, 11,13-dimethylheptacosane, 2-methylheptacosane and 3-methylheptacosane were found in larger amounts in female extracts than in male ones, which suggests their role in male attraction. In addition, a laser vibrometer device was used to detect a male-female substrate-born vibrations pattern during pre-copulatory period. The female vibrational signal was recorded as mp3 and conveyed, in loop using a minishaker, on pear shoots with C. pyri virgin pairs to interfere with the mating by masking the natural communications.
2018
- Mating-type switching in CBS 732T derived subcultures unveils potential genetic and phenotypic novelties in haploid Zygosaccharomyces rouxii.
[Articolo su rivista]
Bizzarri, M; Cassanelli, S; Solieri, L.
abstract
In haploid Saccharomyces cerevisiae, a complex recombination system regulates mating-type switching and requires one MAT expression locus, two donor cassettes (HML and HMR) and the HO endonuclease that catalyses gene conversion. Zygosaccharomyces rouxii is the most distant species from S. cerevisiae with a functional HO, but with a poorly understood mating-type switching. Here, we described that two subcultures of the type strain CBS 732T underwent the α to a genotype switching leading to mixed MATα and MATa populations. Remarkably, during this event the donor cassette was copied into the MAT locus, except for its own 3΄ end, resulting in a new MATa2 gene copy different from the silenced HMRa2. Moreover, CBS 732T cells bypassed the cell-cycle control, which oversees HO transcription in S. cerevisiae, and expressed HO at the stationary phase. Despite HO dysregulation, mating-type switching seemed to occur rarely or belatedly during CBS 732T colony formation in most of the tested conditions. When morphology and mating behaviour were analysed, two subcultures displayed distinct outcross fertility responses. Overall, our data support that mating-type switching causes genotype instability and phenotypic novelties in CBS 732T, and open the question whether this mechanism is shared by other Z. rouxii haploid homothallic strains.
2018
- Mining genes involved in indoxacarb resistance of Lobesia botrana (Denis and Schiffermüller) by de novo transcriptome assembly and differential expression analysis.
[Abstract in Atti di Convegno]
Cassanelli, Stefano; Civolani, Stefano; Salvemini, Marco; Carrieri, Alberto; Bernacchia, Giovanni; Finetti, Luca; Butturini, Alda; Scapoli, Chiara
abstract
Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae) is one of the most important grapevine pests in Europe but, being a non-model organism, only limited genomic and transcriptomic resources are available for functional studies at the molecular level, such as those relevant to insecticide resistance and pest control. Hence, to gain insight into the mechanism of indoxacarb resistance, a blocker of insect voltage-gated sodium channels (NaV), we analysed the transcriptome and expression profile in 2nd instars of L. botrana from susceptible and field selected populations (LC50 resistance ratio 72). De novo transcriptome assembly using Trinity resulted in 141,581 isoforms clustered in 94,290 putative genes. The transcriptome completeness was supported by BUSCO: 92% of conserved orthologs (n= 1,658) were retrieved as a complete sequence, 6.3% displayed fragmented ORFs, and only 1.7% were missing. 36,250 genes were preliminary annotated relaying on the longest isoform per gene, by running Annocript pipeline against non-redundant protein databases (Nr), gene ontology (GO), cluster of orthologous groups of proteins (COG), KEGG orthology (KO) and long non-coding RNAs (lncRNAs). Conditional Reciprocal Best BLAST analysis of protein isoforms performed on Lepidoptera proteomes identified putative orthologs of multigene family members potentially involved in metabolic resistance (61 cytochrome P450 monooxygenases, 25 glutathione S-transferases, 13 carboxylesterases, 25 UDP-glucuronosyltransferases) as well as alternatively spliced isoforms of the NaV gene. Among 263 upregulated and annotated genes in the resistant population, functional GO enrichment analysis revealed overrepresentation of terms for cytochrome P450, due to up-regulation of CYP6B and CYP9A subfamily members as well as increased transcript level for UGT genes. Hydrolases were, on the contrary, overrepresented in 293 annotated genes, downregulated in the resistant population. These data tentatively suggest the reduced susceptibility to indoxacarb might be related to an increase of Phase I and II detoxification along with reduced bioactivation of the insecticide.
2017
- Allodiploid Zygosaccharomyces genomes sequencing assists in deciphering the genetic basis of hybrid sterility
[Abstract in Atti di Convegno]
Bizzarri, M; Pryszcz, L; Solieri, L; Cassanelli, S; Sychrová, H
abstract
In Saccharomyces cerevisiae diploid MATα/MATa cells, a1-α2 heterodimer encoded by mating-type (MAT) genes is the master sensor of diploidy, promoting the meiosis under starvation, and the repression of haploid-specific genes (h-sgs), such as HO gene, under standard growth conditions. However, how other biotech yeasts govern life cycle remains poorly understood. Halotolerant Zygosaccharomyces rouxii yeasts have applications in food spoilage and fermentation, and exhibit ploidy and karyotype variation, tendency to hybridization, and high diversity in osmostress response. Here, the allodiploid Z. rouxii ATCC42981 serves as a model to investigate how transcriptional network incompatibility affects hybrid sterility.
To extend the Zygosaccharomyces genomic knowledge, we sequenced ATCC42981 and Z. sapae ABT301T genomes. We obtained high-quality assemblies (33-45 scaffolds) by combining heterozygosity reduction and assisting the assembling process with Z. rouxii CBS732T reference chromosomes. Both strains are hybrids derived from one lineage that is over 99% identical to CBS732T and another one not yet identified and ~14% diverging from Z. rouxii type-strain. Interestingly, ATCC42981 and ABT301T differ from one another for the second parent. ATCC42981 kept all reference chromosomes, while ABT301T probably lost one. Both genomes harbor chromosomes from the second parental species, suggesting that unexpectedly no reduction of one of the parental karyotypes occurred after hybridization to resolve genome incompatibility.In ATCC42981 genome, MATa1 and MATα2 genes are from two different parents (Z. rouxii and Z. sapae, respectively). The different evolutionary history of a1 and α2 subunits could generate negative epistasis accounting for ATCC42981 inability to repress HO gene and to regulate the main meiosis inducer IME4 gene. To prove this, Z. sapae MATα was disrupted by a loxP–kanMX–loxP cassette. Surprisingly, we found that the deletion mutants still actively transcribed MATα, expressed HO and were unable to sporulate by autodiploidization. Besides, MATα removal did not rescue the ability of ATCC42981 to make conjugated asci in mixture with Z. rouxii mating testers. Overall, this work suggests that 1) HO expression does not assure mating-type switching; 2) with 1 copy of MAT disrupted, ATCC42981 does not behave as a haploid; 3) MATα deletion induces HMLα loci de-silencing or, alternatively, reveals the incomplete silencing of donor cassettes in the wild type strain.
2017
- Drosophila, efficaci le reti ma servono soluzioni più convenienti
[Articolo su rivista]
Caruso, Stefano; Vaccari, Giacomo; Lugli, Stefano; Bellelli, Sara; Francati, Santolo; Maistrello, Lara; Gullo, Maria; Civolani, Stefano; Cassanelli, Stefano
abstract
In attesa di individuare tecniche di controllo biologico e biotecnologico efficacemente integrate con la difesa chimica, le reti multifunzionali rappresentano un sistema collaudato e trasferibile alla produzione, ancorché molto oneroso, di non semplice gestione e talora non idoneo per il ceraseto dal punto di vista microclimatico. Tante attività sperimentali in corso, ma, come al solito, manca a livello nazionale un coordinamento strutturato, non volontario, fatto di attività progettuali integrate.
2016
- Chimeric sex-determining chromosomal regions and dysregulation of cell-type identity in a sterile zygosaccharomyces allodiploid yeast
[Articolo su rivista]
Bizzarri, Melissa; Giudici, P.; Cassanelli, S.; Solieri, L.
abstract
Allodiploidization is a fundamental yet evolutionary poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MATexpression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged forMATa2 genes. Each MATexpression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and a coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and a-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid a-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity.
2016
- Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.
[Articolo su rivista]
Solieri, Lisa; Vezzani, Veronica; Cassanelli, Stefano; Dakal, Tikam Chand; Pazzini, Jacopo; Giudici, Paolo
abstract
The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na+/H+ antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na+/H+ antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301T (low salt tolerant), Z. rouxii CBS 732T (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732T mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.
2016
- Recent advances in understanding yeast genetics of sex determination.
[Articolo su rivista]
Cassanelli, Stefano; Bizzarri, Melissa; Solieri, Lisa
abstract
Sex determination is among the most fascinating areas of study in
modern genetics and encompasses many topics, such as developmental
mechanisms, behaviour, sex chromosome biology, population
evolution and diversity. Yeasts from the subphylum Saccharomycotina
are of great importance to humans, not only as pathogens but for
numerous essential ecosystem services and serve as model to study
evolution in action. The recent advent of inexpensive sequence
information and other new tools has led to notable advances in our
understanding of reproductive mechanisms over a broad range of
species, revealing that genetic sex determination occurs in different
ways with a myriad of outcomes in yeast.
2015
- A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance
[Articolo su rivista]
Cassanelli, Stefano; Ahmad, Shakeel; Duso, Carlo; Tirello, Paola; Pozzebon, Alberto
abstract
The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as
organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based onpredatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been
reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae),
resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and
prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant
strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same
mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping
can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).
2015
- An integrated approach to achieve environmentally sustainable control of the new emerging pest Drosophila suzukii in cherry orchards of
Emilia Romagna
[Poster]
Civolani, S.; Cassanelli, Stefano; Dindo, M.; Fano, E.; Francati, S.; Gullo, Maria; Maini, S.; Maistrello, Lara; Marchetti, E.; Lugli, S.; Vaccari, Giacomo; Caruso, S.
abstract
An integrated approach is presented in this study for achieving environmentally sustainable control of Spotted Wing Drosophila (SWD), Drosophila suzukii, in cherry orchards of the Emilia-Romagna region. Analysis was conducted on the response of SWD based on a variety of control strategies and techniques in laboratory and field conditions. The mechanical control considered the use of net covers to avoid pest infestation. Field monitoring was performed to investigate SWD flight patterns and infestations. The biological control of the pest considered the use of native natural enemies predators and parasitoids).
Entomo-pathogenic microorganisms were also investigated in the context of microbiological
control. The aim is to enhance SWD self-infection using a ‘catch and release’ traps, which
attract the adults by means of appropriate chemical and physical stimuli, exposing them to
entomo-pathogenic microorganisms. Finally, toxicological (LC50 and LC90) and enzymatic
bioassays were performed using different insecticides on different populations to assess
SWD resistance. The results of the study will be useful for a better control of this invasive
insect following integrated pest management and protecting native beneficial insects.
2015
- Ectopic recombination in sex-determination system as a source of genetic variation in the diploid yeast Zygosaccharomyces sapae.
[Abstract in Atti di Convegno]
Bizzarri, Melissa; Verspohl, Alexandra; Giudici, Paolo; Cassanelli, Stefano; Solieri, Lisa
abstract
Sexual reproduction increases genetic variation, which is strongly advantageous under harsh environmental conditions, as it allows natural selection to proceed more effectively. The yeasts of the Zygosaccharomyces rouxii complex are relevant in food elaboration and spoilage due to their ability to cope with low water activity environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy (Solieri et al. 2013). The mating-type locus (MAT) is a hotspot for chromosome rearrangement in yeasts. Here, we investigated the genetic architecture of sex determinants, including MAT loci and HO endonuclease in Zygosaccharomyces sapae diploid strain ABT301T, belonging to the Z. rouxii complex. We cloned these genes through a DNA walking strategy and a characterization of the flanking regions, while the chromosome assignment was performed combining Southern blotting and PFGE-karyotyping. We identified three divergent mating type-like (MTL) α-idiomorph sequences, designated as ZsMTLα copies 1, 2, and 3, which encoded homologues of Z. rouxii CBS 732T MATα2 (aa sequence identity from 67.0 to 99.5%) and MATα1 (identity 81.5-99.5%). Cloning of MATa-idiomorph yielded one ZsMTLa locus encoding two Z. rouxii-like proteins, MATa1 and MATa2. ABT301T possesses two divergent HO genes encoding distinct endonucleases. Based on the cloned ZsMTLα and ZsMTLa idiomorphs flanking regions we discovered that Z. sapae ABT301T displays an aααα genotype lacking the HMR silent cassette. Additionally, four putative HML cassettes were identified, two harbouring the ZsMTLα copy 1 and the remaining containing ZsMTLα copies 2 and 3. In conclusion, our results show that the mating-type switching is responsible for hyper-mutation in Z. rouxii complex. The ectopic recombination underlying this process is an error-prone mechanism, which represents a possible source of genetic variation providing yeast progeny with phenotypic variability and adaptation to hostile environments.
2015
- Testing Spirotetramat as an Alternative Solution to Abamectin for Cacopsylla pyri (Hemiptera: Psyllidae) Control: Laboratory and Field Tests
[Articolo su rivista]
Civolani, Stefano; Boselli, Mauro; Butturini, Alda; Chicca, Milvia; Cassanelli, Stefano; Tommasini, Maria Grazia; Aschonitis, Vassilis; Fano, Elisa Anna
abstract
Aim of the study was to investigate the performance of the new insecticide "spirotetramat" as an alternative solution of "abamectin" for the control of Cacopsylla pyri L. (Hemiptera: Psyllidae) in the context of an IPM program in European pear, Pyrus communis L.. Laboratory bioassays for the estimation of LC50 and LC90 of both insecticides were performed using four populations collected in Emilia-Romagna (Italy) orchards where different pest management strategies were used (organic, integrated, and conventional). The same populations were also analyzed for the main insecticide detoxifying activities in nymphs by spectrofluorimetric in vitro assays. The performance of the two insecticides was also tested on field on one population under integrated pest management conditions. The laboratory experiments showed that the LC90 of spirotetramat were lower than the highest field concentration allowed in Europe (172.80 mg AI liter(-1)) giving reassurance about the efficacy of the product. Concerning the abamectin, the laboratory bioassays did not show strong indications of resistance development of C. pyri populations of Emilia-Romagna. A similarity in enzyme detoxifying activity was observed in both insecticides indicating a general absence of a significant insecticide resistance. The field trial showed a high efficacy (>90 %) of spirotetramat on C. pyri already after 15 d from application, and it was significantly higher from abamectin. Overall, spirotetramat is one more choice for C. pyri control, as well as abamectin in order to minimize the risks of occurrence of insecticide resistance.
2015
- The sex determination system and the role of chimeric a1/alpha2 heterodimer in the sterility of an allodiploid Zygosaccharomyces yeast
[Abstract in Rivista]
Solieri, Lisa; Bizzarri, Melissa; Giudici, Paolo; Cassanelli, Stefano
abstract
In Saccharomyces cerevisiae diploids, the a1/alpha2 protein heterodimer acts as repressor of haploid-specific
genes, such as MATalpha1 and HO genes, and allows a/alpha diploid cells to undergo sporulation. The regulatory
routes governing the cell type and fate have been not yet studied in the yeasts belonging to the
Zygosaccharomyces rouxii complex. These yeasts are relevant in foodstuff elaboration and spoilage due to a
wide repertoire of tolerances to osmotic stress. Their genetic variability favors phenotypic diversity and
adaptation to hostile environments by ectopic recombination of mating-type (MTL) loci through an error-prone
switching mechanism. Among them, the allodiploid strain ATCC42981 exhibits hybrid vigor and multi-stress
tolerance, but it is unable to undergo sexual reproduction. Aim of this work was to characterize allodiploid strain
ATCC42981 for the genetic organization and the transcriptional expression of mating type-like (MTL) loci and
HO genes. Genetic dissection of ATCC42981 sex determinants revealed a MATa/MATalpha genotype with a
redundant number of partial divergent HMR cassettes. MATa expression locus contained Z. rouxii-related MATa1
and MATa2 genes, whereas MATalpha expression locus contained Z. sapae-related MATalpha1 and MATalpha2
genes (termed MATalpha1 and MATalpha2 copy 2, respectively). Both MAT expression loci are linked to
phylogenetically congruent HML silent cassettes on non-homologous chromosomes. Other than a “hybrid” and
redundant three-cassette system, ATCC42981 possesses two divergent HO genes. Consistently with this gene
organization, ATCC42981 expressed MATa1 and MATalpha2 copy 2 genes under standard conditions, whereas aand
α-idiomorph genes from HMR and HML cassettes were silent. Differently from S. cerevisiae diploids,
ATCC42981 did not repress either MATalpha1 or HO gene transcription. Under hypersaline stress (which should
be induce meiosis in Zygosaccharomyces cells by turning on the haploid gene-specific program), ATCC42981
was not able to undergo meiosis and over-expressed HO copy 2, MATa1 and MATalpha1 copy 2. We
hypothesized that the partial incompatibility between ATCC42981 Z. rouxii-like a1 and Z. sapae-like alpha2
subunits in MATa1/alpha2 heterodimer causes a defective silencing of haploid-specific genes, including meiosis
inhibiting factors, leading to clonality as the only possible reproduction strategy for this allodiploid yeast
2014
- An EPG study of the probing behavior of adult Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae) following exposure to cyantraniliprole
[Articolo su rivista]
Civolani, Stefano; Cassanelli, Stefano; Chicca, Milvia; Rison, Jean L; Bassi, Andrea; Alvarez, Juan M; Annan, I. Billy; Parrella, Giuseppe; Giorgini, Massimo; Fano, Elisa Anna
abstract
Cyantraniliprole is a novel insecticide for control of multiple chewing and sucking insect pest species including the sweetpotato whitefly Bemisia tabaci (Gennadius), which is one of the most important polyphagous pests in tropical, subtropical, and Mediterranean regions. This study aims to evaluate the effects of cyantraniliprole on the probing behavior of B. tabaci on tomato. Electrical penetration graph data indicated that on plants treated with cyantraniliprole (foliar application), adult whiteflies of the genetic variant Q2 were not able to reach the phloem and consequently did not perform the activities represented by E1 and E2 waveforms, i.e., phloem salivation (during which inoculation of geminiviruses occurs) and phloem sap ingestion (during which geminiviruses are acquired by the whiteflies), respectively. The complete failure of B. tabaci biotype Q adults to feed from the phloem of tomato plants treated with cyantraniliprole could be explained by rapid cessation of ingestion because of the mode of action of this insecticide. Overall, these findings indicated that cyantraniliprole might represent a useful new tool for producers to protect tomato plants from damage by B. tabaci.
2014
- Assessment of insecticide resistance of Lobesia botrana (Lepidoptera: Tortricidae) in Emilia-Romagna region
[Articolo su rivista]
Civolani, Stefano; Boselli, Mauro; Butturini, Alda; Chicca, Milvia; Fano, Elisa Anna; Cassanelli, Stefano
abstract
The European grapevine moth, Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae), is the key pest of vineyard, Vitis vinifera L. In Italy, failures in field chemical pest control have been recently reported. The susceptibility to insecticides indoxacarb, methoxyfenozide, and emamectin benzoate was then evaluated in a L. botrana population collected from a vineyard in Emilia-Romagna (northeastern Italy) where pest management programs achieved unsatisfactory results. The field trial showed that the indoxacarb efficacy toward L. botrana was very low in the two timings of application (7.9 and -1.5%) in comparison with untreated control, while the efficacy of methoxyfenozide (76.1%) and emamectin benzoate (88.8%) was high. The decreased efficacy of indoxacarb was also supported by the results of the laboratory bioassay on neonate L. botrana larvae, in which the resistance ratio was 72-fold in comparison with that of the susceptible strain.
2014
- Resistenza ad acaricidi e acaricidi
[Capitolo/Saggio]
Cassanelli, Stefano; Butturini, Alda
abstract
Nel libro gli autori, sulla base dei principi e criteri che definiscono la difesa integrata (misure preventive, giustificazione degli interventi, mezzi di difesa a basso impatto, razionalizzazione nell’impiego dei prodotti fitosanitari) indicato le soluzioni e le tecniche attualmente applicabili e derivanti da pluriennale esperienza. L’obiettivo è unire la salvaguardia delle risorse ambientali alla difesa della salute valorizzando la produzione, nel rispetto della direttiva n. 128/09/UE sull’uso sostenibile dei prodotti fitosanitari e dei principi generali della difesa integrata. L’opera rientra a pieno titolo quale supporto informativo per tecnici e agricoltori che devono applicare soluzioni tecniche coerenti con gli obiettivi di un’agricoltura sostenibile.
2014
- Sex-determination system in the diploid yeast Zygosaccharomyces sapae
[Articolo su rivista]
Solieri, Lisa; Dakal, TIKAM CHAND; Giudici, Paolo; Cassanelli, Stefano
abstract
Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments.
2014
- Studio dei semiochimici intraspecifici ed interspecifici per il controllo di Cacopsylla pyri (L.).
[Abstract in Atti di Convegno]
Ganassi, Sonia; Germinara, G. S. .; Santo, P. Di; Pati, S.; Civolani, S.; Cassanelli, Stefano; Rama, F.; Sabatini, Maria Agnese; Cristofaro, A. De
abstract
Cacopsylla pyri (L.) (Hemiptera, Psyllidae) è uno degli insetti più dannosi al pero; il suo mancato controllo provoca significative riduzioni quali-quantitative della produzione. Cacopsylla pyri produce melata che causa sviluppo di fumaggini, necrosi di tessuti fogliari e frutti, ed è responsabile della trasmissione del fitoplasma “Candidatus Phytoplasma
pyri’’. Il contenimento del fitomizo è spesso insoddisfacente, soprattutto in assenza del suo predatore naturale, Anthocoris nemoralis. Al fine di promuovere nuove strategie di controllo biotecnico, efficaci e di minor impatto ambientale, sono stati intrapresi studi elettrofisiologici e comportamentali per valutare l’attività biologica di sostanze presenti
negli estratti cuticolari di adulti di C. pyri e di composti volatili di origine vegetale emessi da diverse cultivar di pero..Le risposte EAG hanno evidenziato che i composti volatili presenti negli estratti di femmine e maschi di C. pyri, somministrati a 3 dosi crescenti, stimolano le antenne di entrambi i sessi, anche se con diversa intensità.L’analisi gascromatografica abbinata a spettrometria di massa (GC-MS) su estratti cuticolari di femmine e maschi ha permesso l’identificazione, tra gli altri, di quattro composti alifatici a lunga catena.I quattro composti identificati, presenti in quantità maggiori nella cuticola delle femmine rispetto a quella dei maschi, sono i più probabili candidati quali componenti della miscela feromonica di C. pyri.Tra i tanti composti volatili emessi dalle diverse cv di pero, tutti quelli saggiati sulle antenne di femmine e maschi di C. pyri hanno evocato risposte EAG evidenti quantificabili; tali risposte, generalmente maggiori nelle femmine, sono risultate significativamente più elevate solo per alcune aldeidi . Tali composti potrebbero essere utilizzati, ad esempio, nel monitoraggio delle femmine di C. pyri, per modificarne il comportamento nella scelta della pianta ospite, del sito di ovideposizione, o per potenziare l’attrattività del feromone sessuale.
2014
- Studio per una strategia innovativa per il controllo biotecnico di Cacopsylla pyri (L.) (Hemiptera, Psyllidae)
[Abstract in Atti di Convegno]
Civolani, S.; Ganassi, Sonia; Germinara, G. S; Santo, P. Di; Pati, S.; Fano, E. A.; Cassanelli, Stefano; Sabatini, Maria Agnese; Rama, F.; Cristofaro, A. De
abstract
Nella moderna difesa delle coltivazioni agricole vi è l’esigenza, raccomandata dall’Unione Europea, di implementare tutte le tecniche innovative di controllo delle avversità che limitino l’impiego di fitofarmaci in un contesto di agricoltura sostenibile. E’ stato pertanto intrapreso uno studio per valutare l’uso di composti volatili (feromoni sessuali e cairomoni), nel controllo di Cacopsylla pyri (L.) uno dei fitofagi più dannosi al pero. L’utilizzo dei semiochimici, pratica consolidata nel controllo di fitofagi, esteso alla difesa del pero da C. pyri può condurre a una diminuzione delle popolazioni del fitomizio e dell’impiego di prodotti fitosanitari, con conseguente aumento quali-quantitativo della produzione; ciò avrebbe un’indubbia ricaduta positiva sull’attività economica e sulla sostenibilità dell’azienda agricola, con beneficio economico ed ambientale. La combinazione di indagini elettroantennografiche e olfattometriche ha evidenziato la presenza di feromone sessuale nella cuticola delle femmine. Le analisi GC-MS, infatti, hanno indicato che 13-metileptacosano, 11,13-dimetileptacosano, 2-metileptacosano e 3-metileptacosano, ottenuti in quantità maggiori (da 3 a 14 volte) da estratti cuticolari delle femmine sono i più probabili candidati quali componenti della miscela feromonica di C. pyri. 13-metileptacosano è stato oggetto di sintesi chimica per valutarne in campo l’attività attrattiva sui maschi.
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
- MONITORAGGIO DELLO SVILUPPO DELLA RESISTENZA AGLI INSETTICIDI NEL LEPIDOTTERO LOBESIA BOTRANA
[Poster]
Cassanelli, Stefano; Casali, Giulia; Pagni, MARIA ROSA; Garcia, Loretto; Pradolesi, Gianfranco; Butturini, Alda; Civolani, Stefano
abstract
La Tignoletta della vite, Lobesia botrana (Denis & Schiffermüller, 1775), lepidottero tortricide, è una delle principali avversità entomologiche della vite, a causa dei danni arrecati dalle larve agli acini dell’uva. Il quadro delle sostanze attive più frequentemente utilizzate nella difesa della vite dalla tignoletta comprende attualmente prodotti quali methoxyfenozide e indoxacarb, recentemente affiancati dall’emamectina, chlorantraniliprole e spinosad. Nella produzione integrata la scelta e l’impiego razionale dei mezzi di difesa deve necessariamente tener conto della possibilità che i fitofagi sviluppino progressivamente una ridotta suscettibilità ai prodotti fitosanitari (resistenza). Lo studio si è proposto d’indagare in popolazioni raccolte in Emilia-Romagna: a) la presenza di resistenza alle sostanze attive introdotte da tempo sul mercato; b) la sensibilità alle nuove sostanze attive da poco introdotte nei programmi di difesa integrata c) la presenza di resistenza crociata tra le diverse sostanze attive. Le metodiche di laboratorio utilizzate sono state messe a punto nell’ambito delle attività di ricerca di progetti CRPV finanziati dalla Regione Emilia-Romagna e prevedono l’analisi della sensibilità degli insetti alle singole sostanze attive (biosaggi) e del livello di attività detossificante gli insetticidi (test enzimatici). Le indagini svolte mostrano l’efficacia dei prodotti di più recente introduzione e un calo della tenuta dei principi attivi con uno storico d’uso prolungato, associato ad un aumento delle attività enzimatiche detossificanti GST ed MFO. In conclusione lo studio ha rivelato l’esistenza di un progressivo adattamento metabolico in L. botrana come risposta ai trattamenti con i principi attivi d’utilizzo più datato. Tale adattamento, tuttavia, non consente il contrasto dell’azione insetticida prodotta delle nuove molecole, determinando l’assenza di resistenza crociata fra i prodotti con una diversa tempistica d’inserimento nei disciplinari di difesa.
2013
- Studio dell’attività biologica di semiochimici per una strategia innovativa di biocontrollo di Cacopsylla pyri (Hemiptera, Psyllidae).
[Abstract in Rivista]
S., Ganassi; P., DI SANTO; S., Civolani; Cassanelli, Stefano; Sabatini, Maria Agnese; A., DE CRISTOFARO
abstract
Cacopsylla pyri è uno dei più importanti insetti dannosi al pero in Emilia Romagna poiché causa una significativa riduzione qualitativa e quantitativa della produzione. Il contenimento del fitofago, basato su un numero limitato di fitofarmaci, è spesso insoddisfacente e con risultati variabili soprattutto quando manca il contributo naturale del suo predatore, Anthocoris nemoralis. E’ stato pertanto intrapreso uno studio per valutare l’attività biologica di semiochimici intraspecifici (feromoni sessuali) ed interspecifici (sostanze volatili di origine vegetale) su C. pyri per una strategia innovativa di biocontrollo di tale fitofago. Lo studio si articola in due linee di indagine; la prima ha previsto l’ottenimento, sia da femmine che da maschi di C. pyri, di estratti cuticolari la cui attività biologica è stata valutata mediante indagini elettroantennografiche (EAG) e comportamentali (olfattometro a Y). Le risposte EAG hanno evidenziato come le miscele di composti volatili presenti negli estratti ottenuti da femmine fossero in grado di stimolare il sistema olfattivo dei maschi. Indagini olfattometriche successive hanno dimostrato come i maschi fossero significativamente attratti sia dalle femmine che dal loro estratto cuticolare, evidenziando la presenza di feromoni sessuali in corso di identificazione. La seconda linea di indagine riguarda l’individuazione di sostanze volatili emesse da diverse cultivar di pero in grado di influenzare il comportamento di C. pyri e di conseguenza la scelta della pianta ospite. Tutte le sostanze volatili saggiate hanno evocato risposte EAG: alcune risposte sono risultate significativamente più elevate per entrambi i sessi.
2013
- Susceptibility to spirotetramat and abamectin of pear psylla Cacopsylla pyri L. (Hemiptera: Psyllidae) in Northern Italy
[Relazione in Atti di Convegno]
Stefano, Civolani; Mauro, Boselli; Alda, Butturini; Cassanelli, Stefano
abstract
Spirotetramat may be considered a valuable tool for the control of C. pyri as a component of an IPM programme. Given the high efficiency on C. pyri populations in field test this active ingredient could represent a valuable alternative to abamectin in order to manage the risks of occurrence of resistance in C. pyri control. Overall, the bioassays data indicate that no apparent resistance to abamectin has yet been developed in C. pyri populations of Emilia-Romagna. Nevertheless, the pear orchards in which C. pyri outbreaks recently occurred are under close investigation and careful survey.
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
- 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
- Resistance to acaricides in Italian strains of Tetranychus urticae: toxicological and enzymatic assays.
[Articolo su rivista]
P., Tirello; A., Pozzebon; Cassanelli, Stefano; T., Van Leeuwen; C., Duso
abstract
Problems with Tetranychus urticae are frequently reported in protected crops in Italy, particularly in roses where many introduced acaricides show a progressive loss of effectiveness. We have conducted bioassays to assess the response of some Italian strains of T. urticae to a number of acaricides. These include compounds that were widespread and frequently used in the past, but also some recently registered compounds. We investigated two T. urticae strains collected from rose growers where control failures were reported (SAN and PSE), together with a strain collected from unsprayed vegetables (BOSA). Adult females of the rose strains (SAN and PSE) were resistant to tebufenpyrad (Resistant Ratio-RR, RR(50) = 48.4 and 163.6) and fenpyroximate (RR(50) = 74.1 and 25.9) when compared to the susceptible BOSA strain. Lethal concentrations for these products were higher than the registered field rate. The PSE strain proved to be highly resistant to abamectin (RR(50) = 1,294.1). Variation in bifenazate susceptibility was detected amongst strains, but LC(90) values of SAN and PSE were still in the range of the registered field rate. In egg bioassays, the SAN and PSE strains exhibited high resistance levels to clofentezine (RR(50) = 66,473 and 170,714), hexythiazox (RR(50) = 70,244 and 159,493) and flufenoxuron (RR(50) = 61.9 and 117.9). But the recently introduced ovi/larvicides etoxazole and spirodiclofen exhibited high activity on all strains. The activity of detoxifying enzymes such as esterases, glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (MFOs) was determined in these strains as a preliminary attempt to identify potential resistance mechanisms. Enzymatic assays showed that the rose strains exhibited 2.66 and 1.95-fold increased MFOs activity compared to the susceptible strain. Assays for GSTs revealed that only the SAN strain exhibited a significantly higher activity. In contrast, only the PSE strain showed a significant higher hydrolysis of 1-naphthyl acetate.
2011
- Combined detoxification mechanisms and target mutation fail to confer a high level of resistance to organophosphates in Cydia pomonella (L.) (Lepidoptera: Tortricidae)
[Articolo su rivista]
Reyes, Maritza; Collange, Béatrice; Rault, Magali; Cassanelli, Stefano; Sauphanor, Benoît
abstract
Despite the frequent and widespread applications of organophosphates against Cydia pomonella this species has developed low levels of resistance to this chemical group. Investigations concerning the mechanisms involved in resistance are scarce, and usually consider only one of the potential mechanisms. With the aim of a better understanding the resistance mechanisms and their possible interaction, four of these mechanisms were investigated simultaneously in one sensitive (Sv) and two resistant strains (Raz and Rdfb) of this insect. Resistant strains displayed an increased mixed function oxidase activity, whereas carboxylesterase activity varied upon the substrate used. The three strains had similar β-naphtyl acetate activity, and the hydrolysis of α-naphthyl acetate and p-nitrophenyl valerate was higher in the Sv strain. The p-nitrophenyl acetate activity was highest in the resistant strains and was strongly inhibited by azinphos and DEF. The Raz strain has a modified acetylcholinesterase (AChE), which resulted in a 0.7-, 3.2- and 21.2-fold decrease in the susceptibility to chlorpyriphos-ethyl-oxon, azinphos-methyl-oxon, and paraoxon-methyl, respectively. These combined resistance mechanisms only conferred to Raz a 0.6-, 7.9- and 3.1-fold resistance to the related insecticides. Organophosphates resistance in C. pomonella results from a combination of mechanisms including modified affinities to carboxylesterase substrates, and increased metabolisation of the insecticide. The apparent antagonism between increased functionalisation and reduced sensitivity of the AChE target is discussed.
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
- 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.
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
- Chromosomal polymorphism and ploidy divergence in Zygosacharomyces rouxii strains isolated from Traditional Balsamic Vinegars
[Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract
The genus Zygosaccharomyces traditionally describes osmophilic yeasts that differ from the those of the genus Saccharomyces for their vegetative growth, in which sexual conjugation between cells or a cell and its bud, precedes the sporulation1. Diploidization through zygote formation means that vegetative life cycle is mainly haploid. In the last past decade phenotypical and molecular approaches based on cloning procedure, single-gene PCR amplification and karyotyping highlighted a high variability within Zygosaccharomyces rouxii species, mainly between type strain CBS 732 and some wild strains isolated from miso and traditional balsamic vinegar (ATCC 42981, ABT 301, and ABT 601, respectively)2,3,4. Recently random fragments sequencing from strain ATCC 42981 and allele-specific PCR reaction from strain ABT301 and ABT 601 have revealed that all these wild strains have two copies of several genes, including ITS-5.8S rDNA, 26S D1/D2 regions, ZSOD and HIS34,5. Genome size is one of the most fundamental genetic properties of living organisms. However no study has been attempted to evaluate genome size and ploidy for Z. rouxii strains. In this work genome size and ploidy level estimation, as well as ZSOD gene chromosome mapping were used to elucidate the taxonomic relationships among these wild strains compared to Z. rouxii CBS 732T.In particular, we applied flow cytometry to determine DNA content of cells in G0/G1 phase. The genome size was estimated 11.89±0.21 Mb (12.1±0.22 fg) for strain CBS 732T, 21.08±0.37 Mb (21.56±0.37 fg) for ATCC 42981, 25.68±1.11 Mb (26.27±1.14) for ABT 301, and 36.00±0.37 Mb (36.80±0.38 fg) for ABT 601. Moreover pulsed field gel electrophoresis (PFGE) confirmed the genome size divergence with polymorphic PFGE patterns consisting in different number of chromosomal bands: 6, 7, 10, and 11 for CBS 732T, ATCC 42981, ABT 301, and ABT 601, respectively. By densitometric analysis of PFGE profile, the haploid genome size has been obtained as sum of chromosomal bands size. These data have been combined to DNA content from flow cytometric analysis in order to calculate the ploidy level for each strain. The results showed CBS 732T has a haploid DNA content, whereas wild strains ATCC 42981, ABT 301, and ABT 601 a diploid DNA content. Finally the change in ploidy has been also confirmed by doubling of genes involved in osmotic adaptation (ZSOD, HOG, GPD, and GCY1), as well as by polymorphic rDNA genes occurring in ATCC 429815,6 and in ABT 301 and ABT 6014, but not in CBS 732T 2. For this purpose chromosome mapping of ZSOD genes (encoding Na+/H+-antiporters involved in halotolerance) showed that, differently from haploid CBS 732T, diploid strains ATCC 42981, ABT 301 and ABT 601 have two partially divergent ZSOD copies located on different chromosomes. In conclusion genome size and ploidy are two fundamental and informative traits mainly for yeasts with complex diploid/haploid shift in their life cycle. Our complementary molecular strategies highlighted a naturally occurring variation in genome size and ploidy level within Z. rouxii, suggesting that there is a complex picture in genome organization and the strains now recognized as Z. rouxii could belong to more than one species. References1. Barker, B.T.P., Trans. R. Soc. Lond. B., 194, 467–485 (1901).2. Kinclova, O., et al.,. J. Biotechnol., 88, 151–158. (2001)3. Pribylova, L., et al., Yeast, 24, 171-180 (2007)4. Solieri, L., et al., Yeast, 24, 403–417 (2007)5. Gordon, J., Wolfe, K.H. Yeast, 25, 449-456 (2008) 6. Iwaki T., et al., Microbiol. 145, 241-248 (1999).
2008
- Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species
[Articolo su rivista]
Solieri, Lisa; Cassanelli, Stefano; Croce, Ma; Giudici, Paolo
abstract
Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.
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.
2007
- A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar.
[Articolo su rivista]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract
The taxonomic status and species number of the genus Zygosaccharomyces have rapidly changed in the last years. In this study, two new osmotolerant Zygosaccharomyces strains isolated from traditional balsamic vinegar, viz. ABT301 and ABT601, were investigated to elucidate their taxonomic relationships with Zygosaccharomyces rouxii species. A multi-gene sequence approach was employed, including regions of the rDNA repeat [5.8S, two internal transcribed spacers (ITS) and the 26S D1/D2 domain], COX2 mitochondrial gene and two nuclear genes (SOD2 and HIS3). Cloning and sequence analysis of 5.8S-ITS rDNA revealed that these strains bear an unusual polymorphism for this region. Three highly divergent 5.8S-ITS sequences were detected, one identical to Z. rouxii, the other two showing some relatedness to Z. mellis. Sequence and gene number polymorphism was also observed for the protein-encoding nuclear genes SOD2 and HIS3, as two copies for each gene different from those found in Z. rouxii were detected. Analysis of the D1/D2 26S domain showed that ABT301 and ABT601 have only one type of D1/D2 sequence statistically different from that of Z. rouxii. The findings obtained in this work suggest that the genomic background of strains ABT301 and ABT601 is different from the other Zygosaccharomyces species. We speculated that they could belong to a new putative species related to Z. rouxii.
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
- Physiological and genotypic characterization of a novel Zygosaccharomyces species
[Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract
A pool of new Zygosaccharomyces strains was isolated in Traditional Balsamic Vinegar samples. Recently an our multi-genestudy [including genes of the rDNA repeat and two nuclear genes, SOD2 and HIS3] has highlighted that: i) rDNA units areunusually polymorphic with respect to ITS regions (with three highly divergent ITS sequences), but the D1/D2 26S rDNA regionis unique and monomorphic; ii) two different copies of nuclear genes SOD2 and HIS3 are present. In this study a physiologicaland genotypic characterization of two of these strains, namely ABT301 and ABT601, was performed. Physiological tests showedthat these strains are osmophilic and halophilic yeasts able to grow at 60% glucose and 16% NaCl and with a assimilative profilepartially similar to that of Z. rouxii species. Chromosomal DNA separation by PFEG was performed in order estimate the numberand size of their chromosomes. A SOD2-22-based Real Time PCR assay was designed to determine the ploidy of ABT301 andABT601, compared to diploid Z. rouxii CBS732 type strain. Finally three transcriptional assays were carried out: the first wasaimed to 5.8S-ITS region of 35S precursor rRNA, the others to the two different SOD2 and HIS3 copies. According to ourpreliminary results, ABT301 and ABT601 showed: i) a different electrophoretic karyotype compared to CBS732T strain; ii) adiploid status; iii) both copies of SOD2 and HIS3 genes actively transcribed, as well as the three 5.8S-ITS regions.
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
- 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
- Molecular dissection of the 5.8S-ITS region variability in Zygosaccharomyces spp.
[Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; S., Rainieri; Giudici, Paolo
abstract
Molecular dissection of the 5.8S-ITS region variability in Zygosaccharomyces spp.
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
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
- Strategies and perspectives for genetic improvement of wine yeasts
[Articolo su rivista]
Giudici, Paolo; Solieri, Lisa; Pulvirenti, Andrea; Cassanelli, Stefano
abstract
Recent developments in expression profile and proteomic techniques illustrated that the main oenological traits of wine yeasts are complex and influenced by several genes, each of them identified as absolutely essential. Only for monogenic properties the genetic improvement programmes of wine yeasts can be performed by alteration of individual genes. Ideally the most productive way of improving the whole-cell biocatalysts is by evolution of the entire cell genome. In this article we briefly review the main genetic improvement techniques applied in new and optimised wine strains construction, paying particular attention to blind and whole genome strategies, such as the sexual recombination and genome shuffling.
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.
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.
2001
- Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene
[Articolo su rivista]
Montosi, Giuliana; Donovan, A.; Totaro, Antonella; Garuti, Cinzia; Pignatti, Elisa; Cassanelli, S.; Trenor, C. C.; Gasparini, P.; Andrews, N. C.; Pietrangelo, Antonello
abstract
Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.
2001
- Frequency and biochemical expression of C282Y/H63D hemochromatosis (HFE) gene mutations in the healthy adult population in Italy
[Articolo su rivista]
Cassanelli, S; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Mariano, M; Campioli, D; Carbonieri, A; Baldini, Enrica; Pietrangelo, Antonello
abstract
Background/Aims: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. Methods: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. Results: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. Conclusions: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
1999
- Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia
[Articolo su rivista]
S., Bertolini; Cassanelli, Stefano; R., Garuti; Ghisellini, Margherita; Ml, Simone; M., Rolleri; CALANDRA BUONAURA, Sebastiano; P., Masturzo
abstract
The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996, The age of the patients ranged from 1 to 64 years; one third of them were older than 30, Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L, The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r = -0.665; P < 0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (less than or equal to 5% of normal) and the highest plasma LDL cholesterol levels. Twenty-nine patients (23 of whom were unrelated) were found to be homozygotes at the LDL receptor locus. In this group we discovered 2 major rearrangements and 12 different point mutations (9 in the coding region and 3 in splice sites). Some mutations (D200G, C358R, V502M, G528D, and P664L) were found in 3 or more unrelated patients. Patients with the same mutation shared the same haplotype at the LDL receptor gene locus and came from the same geographic area. Ten patients (9 of whom were unrelated) were found to be compound heterozygotes. The mutations found in this group consisted of one large deletion and 12 point mutations (11 in the coding sequence and one in a splice site). In 3 compound heterozygotes we failed to identify the second mutant allele at the LDL receptor locus. These observations confirm the allelic heterogeneity underlying familial hypercholesterolemia in the Italian population and indicate that the variability of phenotypic expression of homozygous familial hypercholesterolemia is, to a large extent, related to the type of mutation of the LDL receptor gene.
1999
- Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene
[Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; A., Totaro; Garuti, Cinzia; D., Conte; Cassanelli, Stefano; M., Fraquelli; C., Sardini; F., Vasta; P., Gasparini
abstract
Background and Methods Hereditary hemochromatosis in adults is usually characterized by mutations in the hemochromatosis (HFE) gene on the short arm of chromosome 6. Most patients have a substitution of tyrosine for cysteine at position 282 (C282Y). We studied a large family from Italy that includes persons who have a hereditary iron-overload condition indistinguishable from hemochromatosis but without apparent pathogenic mutations in the HFE gene. We performed biochemical, histologic, and genetic studies of 53 living members of the family, including microsatellite analysis of chromosome 6 and direct sequencing of the HFE gene, Results Of the 53 family members, 15 had abnormal serum ferritin levels, values for transferrin saturation that were higher than 50 percent, or both. Thirteen of the 15 had elevated body iron levels, diagnosed on the basis of the clinical evaluation and liver biopsy, and underwent iron-removal therapy. The other two, both children, did not undergo liver biopsy or iron-removal therapy. None of the 15 members had the C282Y mutation of the HFE gene; 5 of the 15 (as well as 5 healthy relatives) had another mutation of this gene, a substitution of aspartate for histidine at position 63, but none were homozygous for it. No other mutations were found after sequencing of the entire HFE gene for all family members. Microsatellite analysis showed no linkage of the hemochromatosis phenotype with the short arm of chromosome 6, the site of the HFE gene. Conclusions Hereditary hemochromatosis can occur in adults who do not have pathogenic mutations in the hemochromatosis gene. (N Engl J Med 1999;341:725-32,) (C)1999, Massachusetts Medical Society.
1998
- A 'de novo' point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia
[Articolo su rivista]
Cassanelli, Stefano; S., Bertolini; M., Rolleri; F., De Stefano; L., Casarino; N., Elicio; A., Naselli; CALANDRA BUONAURA, Sebastiano
abstract
Severe hypercholesterolemia was found in an Ii-year-old boy with no family history of familial hypercholesterolemia. The reduced LDL-receptor activity in cultured skill fibroblasts (40% I-125-LDL degradation as compared with a control cell line) indicated the presence of an LDL-receptor defect. The analysis of the promoter region and the exons of LDL-receptor gene by single strand conformation polymorphism revealed an abnormal migration pattern in exon 1, which was due to a T-->A transversion at nucleotide 28 of the cDNA. This novel mutation causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) and introduces an AviII restriction site in exon 1. Screening of the mutation by polymerase chain reaction (PCR) amplification of exon 1 and AviII digestion revealed that none of the proband's family members carried the mutation. Non-paternity was excluded after the analysis of a battery of 14 short tandem repeats located in 13 different chromosomes. These results are consistent with the hypothesis that the proband is heterozygous for a 'de novo' mutation of the LDL-receptor gene producing a non-conservative amino acid substitution. We suggest that the change in the net charge of the signal peptide, caused by the addition of a positively charged amino acid, impairs the co-translational translocation of the nascent receptor protein across the endoplasmic reticulum membrane.
1995
- Cytokines and invertebrate immune responses
[Articolo su rivista]
Ottaviani, Enzo; Franchini, Antonella; Cassanelli, Stefano; Genedani, Susanna
abstract
A profound interrelationship between cytokines and invertebrate (molluscs) immune responses has been reported. Different cytokines (IL-1 alpha, IL-2 and TNF-alpha significantly stimulate molluscan hemocyte motility, increasing phagocytic activity and provoking the induction of nitric oxide synthase. As far as cell motility is concerned, the response to different cytokines varied between species. These and other recently reported findings (Ottaviani et al (1994) FEES Lett 351, 19-21; Ottaviani et al (1995) Biochem Biophys Res Commun 207, 288-292) suggest that cytokines are important, ancestral, and functionally conserved molecules, which have also maintained their pleiotropicity, redundancy in the mode of action, and high promiscuity of their receptors during evolution.