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Berardi, Laura (2015) Genetic and proteomic approach to the urticating system of processionary moths (Thaumetopoeinae, Lepidoptera). [Tesi di dottorato]

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Abstract (inglese)

Summary
Larvae of the Thaumetopoea genus produce urticating setae in the third and later instars on the dorsal part of the abdomen in specific areas called mirrors. These setae are easily removed from the integument and are considered to be important for defense against vertebrate predators. Impacts on humans are well described and consist mainly of dermatitis due to contact with the setae and allergic responses. As the mechanism of action in the human skin involves both the mechanical injury of the penetration and the release of proteins with antigenic power, it is important to explore the genes putatively involved in the defense mechanism of the Thaumetopoea species, in order to identify the proteins associated with the setae and elucidate their expression pathway during the larval development. In addition, as the genus includes several species, the variability occurring within and among species should be also explored.
In the introduction of the thesis I present the different type of urticating hairs of the arthropods with a focus on the true setae of the processionary moths, the medical aspects related to them, the epidemiology, and the genes encoding for the antigenic proteins. One of the main aim of my work was to characterize all the proteins, urticating and not, occurring in the setae and try to extract other proteins similar to the main one described so far, Tha p 2. Other goals were to focalize on the expression profile of Tha p 1 (a chemosensory protein of Th. pityocampa) and Tha p 2 in every life stage of Thaumetopoea pityocampa and, at the end, to characterize the orthologous counterparts of Tha p 2 in all the Thaumetopoea species available, as well as in other species of Thaumetopoeinae, to better understand the evolution of the protein in this group.
In the first study I tested two different protein extraction protocols of different strength, in order to create a complete data set of all proteins, urticating and not-urticating, occurring in the setae. Candidate antigenic proteins were recognised by using the sera of persons previously exposed to the agent and showing acute reactions. A high quantity of protein was obtained, confirming that the urticating setae of Th. pityocampa contain proteins, some of which were recognized by Ig-E of persons previously exposed to the larvae of this insect. In addition, I added information about the type, quality and quantity of the proteins associated with the setae.
In the second study I investigated the expression values of the urticating protein Tha p 2 and also of a chemosensory protein Tha p 1, for all life stages of two Italian populations and for the last development stages and eggs of two Portuguese populations, in order to better understand the expression of the gene Tha p 2. I confirmed the expression of Tha p 1 gene in all development stages of Th. pityocampa of all populations, instead the gene Tha p 2 was typically expressed only in those larval instars when the setae are produced, i.e. from the third to the fifth.
In the third study, I sequenced the Tha p 2 gene in all Thaumetopoeinae species available, with a particular focus on members of the genus Thaumetopoea, as well as unrelated moth species, for better understanding the chemical-physical properties of the proteins and the nature of the encoding genes, as well as their evolutionary history. This study permitted to identify two isoforms of Tha p 2 in all species that can be interpreted as the result of heterozygosity in the single gene. The only exception is represented by one species (Thaumetopoea wilkinsoni), in which 20 different isoforms occur in a single specimen, leading to the conclusion that at least in the species multiple copies of Tha p 2 exist.
The Supplementary chapters includes two parts which I developed during the thesis in order to obtain the material used in the analyses. The first concerns the comparison of the developmental time of four populations of the pine processionary moth under controlled laboratory conditions. The four populations, which are characterized by different life history phenology in the field, maintained an annual life cycle also under favorable laboratory conditions, mainly because of a flexible duration of the pupal stage. The second part of the Supplementary chapters is a description of the life history of a species (Thaumetopoea herculeana) collected and studied in Spain for the analysis of the Tha p 2 gene..
On the whole, the thesis is deepening the knowledge on the urticating system of the processionary moths, pointing at identifying the further steps required for the clarification of the complex mechanism associated with the development of reactions in humans and possibly in the vertebrate predators which are the natural target of the setae. In addition, the analyses reveal that the urticating seta system, and the genes associated, are well conserved in the group and could be a major factor affecting their evolution, as well as that of other groups of arthropods which share similar defense mechanisms.



Abstract (italiano)

Riassunto
Le larve delle processionarie europee e mediterranee (genere Thaumetopoea) producono delle setole urticanti dal terzo stadio larvale al quinto sulla parte dorsale dell’addome, in specifiche aree denominate “specchi”. Le setole sono facilmente removibili dal tegumento e sono importanti per la difesa dell’insetto nei confronti di vertebrati predatori. L’impatto delle setole urticanti sull’uomo è stato ripetutamente descritto e consiste prevalentemente nella manifestazione da contatto con la complicazione dell’insorgenza di risposte allergiche. Il meccanismo di azione è complesso e riguarda sia una lesione meccanica causata dalla penetrazione della setola sia il rilascio di proteine con potere antigenico. Risulta quindi importante esplorare i geni potenzialmente coinvolti nel meccanismo di difesa, al fine di identificare le proteine associate con le setole e chiarire la loro espressione durante lo sviluppo larvale. Inoltre, il genere comprende diverse specie e sembra opportuno esplorare la variabilità dei geni coinvolti a livello intra- e interspecifico.
Nell’introduzione della tesi presento i diversi tipi di setole urticanti degli artropodi, focalizzandomi su quelle che vengono denominate “setole urticanti” caratteristiche delle processionarie, gli aspetti medici correlati, l’epidemiologia e i geni correlati alle proteine antigeniche. Uno dei principali obiettivi del mio lavoro è quello di caratterizzare tutte le proteine, urticanti e non, presenti all’interno delle setole e provare a identificare altre proteine antigeniche, oltre alla già nota Tha p 2. Altri obiettivi sono quelli di concentrarsi sul profilo di espressione della proteina Tha p 1 (una proteina isolata da larve di processionaria del pino e riconosciuta come antigene da persone esposte all’insetto, ma successivamente identificata come appartenente al gruppo delle proteine chemosensoriali) e Tha p 2 in tutti gli stadi vitali di Thaumetopoea pityocampa, e la caratterizzazione del gene Tha p 2 in tutte le specie del genere Thaumetopoea disponibili e anche in altre della sottofamiglia delle Thaumetopoeainae, per studiare l’evoluzione della proteina in questo gruppo.
Nel primo studio ho provato due protocolli di estrazione proteica per creare un data set completo di tutte le proteine, sia urticanti che non, presenti nelle setole. Possibili proteine antigeniche sono state riconosciute utilizzando i sieri di persone precedentemente esposte e con reazioni acute. É stata ottenuta un’elevata quantità di proteine che ha permesso di confermare che le setole urticanti di Th. pityocampa contengono proteine, alcune delle quali sono riconosciute da Ig-E di persone precedentemente esposte a larve di questo insetto. Inoltre ho potuto ottenere informazioni riguardo la qualità e la quantità delle proteine associate alle setole.
Nel secondo studio mi sono concentrata sull’espressione dei geni associati alle proteine urticanti Tha p 2 e Tha p 1 in tutti gli stadi larvali di due popolazioni italiane e per gli ultimi stadi e le uova di due popolazioni portoghesi. Ho potuto confermare l’espressione del gene Tha p 1 in tutti gli stadi di Th. pityocampa di tutte le popolazioni, mentre il gene Tha p 2 è espresso solo negli stadi larvali dove le setole sono prodotte (dal terzo al quinto).
Nel terzo studio, ho sequenziato il gene Tha p 2 in tutte le specie della sottofamiglia Thaumetopoeinae disponibili, concentrandomi su membri del genere Thaumetopoea, così come su specie non affini, per capire le proprietà chimico-fisiche della proteina, la natura dei geni e la loro storia evolutiva. Questo studio ha permesso di identificare due diverse isoforme del gene Tha p 2 in tutte le specie, che può essere interpretato come un risultato di eterozigosi del singolo gene. L’unica eccezione è rappresentata da una specie (Thaumetopoea wilkinsoni) nella quale sono state individuate 20 diverse isoforme in un unico campione; ciò porta a pensare che possano esistere molteplici copie del gene.
I capitoli supplementari includono due parti alle quali ho lavorato durante il periodo della tesi, per ottenere materiale utilizzabile durante gli altri esperimenti. La prima parte riguarda il confronto del tempo di sviluppo di quattro popolazioni di processionaria del pino mantenute in condizioni controllate di laboratorio. Le quattro popolazioni, caratterizzate da diversa fenologia, mantengono un ciclo vitale annuale anche in condizioni favorevoli a un rapido sviluppo larvale, soprattutto grazie alla flessibilità della durata dello stadio pupale. La seconda parte dell’appendice è una descrizione della biologia di Thaumetopoea herculeana campionata in Spagna e studiata per l’analisi del gene Tha p 2.
Nel complesso, la tesi approfondisce la conoscenza del sistema urticante delle processionarie, puntando a individuare gli ulteriori passi necessari per chiarire il complesso meccanismo associato allo sviluppo di reazioni cutanee negli esseri umani e, possibilmente, in vertebrati predatori che sono il bersaglio naturale della setole. Inoltre, le analisi rivelano che il sistema urticante e i geni associati sono ben conservati nel gruppo e potrebbero essere un fattore importante nella storia evolutiva in questo e in altri gruppi di artropodi che condividono meccanismi di difesa simili.


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Tipo di EPrint:Tesi di dottorato
Relatore:Battisti, Andrea
Dottorato (corsi e scuole):Ciclo 27 > scuole 27 > SCIENZE DELLE PRODUZIONI VEGETALI
Data di deposito della tesi:27 Gennaio 2015
Anno di Pubblicazione:27 Gennaio 2015
Parole chiave (italiano / inglese):Th. pityocampa, Tha p 2 protein extraction, DNA, RNA seq
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/11 Entomologia generale e applicata
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:7615
Depositato il:13 Nov 2015 13:43
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