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Poltronieri, Carlo (2009) Heat Shock Protein 70 e Rodlet Cells come possibili indicatori di stress in specie ittiche comunemente allevate. [Ph.D. thesis]

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Abstract (english)

The aim of this work was to evaluate the opportunity of using “heat shock protein 70” (Hsp70) and rodlet cells (RCs) as useful parameters to assess stressing conditions in fishes, in particular on farmed fishes. In the first part of the work we evaluate the expression of inducible Hsp70 and the localization of the protein in its inducible and constitutive (Hsc70) form in larvae and juveniles of sea bass (Dicentrarchus labrax) submitted to heat stress condition. This study was performed on both diploid and triploid (of the same age) animals. Our RT-PCR results confirmed, for both diploid and triploid, the expression of the inducible form of Hsp70 after heat shock. The immuno-localization of Hsc70 (constitutive) confirmed, in both diploid and triploid, the ubiquitarious presence of this protein since the first stages of development, regardless of stressing conditions, in different tissues, mainly of epithelial kind (skin, pharynx and gut). Curiously, no immunopositivity was found about the inducible form of Hsp70, in contrast with RT-PCR results.
Successively, we examined the expression of Hsp70 and the localization of both proteins, Hsp70 and Hsc70, in larvae, fry and adults of sea bass before and after transport. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals.
In the present work we, also, investigated the cellular localization of constitutive as well as inducible heat shock protein 70 in several tissues of common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) exposed to transport stress. In carp, the constitutive form (HSC70) was detected only in red skeletal muscle of both control and stressed animals. In the same species, the inducible form (HSP70) was evident in the epithelia of renal tubules, gills and skin of stressed animals, whereas in controls only red skeletal muscle exhibited an immunopositivity to HSP70 antibody. In trout, immunostaining to HSC70 antibody was found mainly in the epithelia of intestine, gills and skin of both control and stressed animals although the reactivity was generally higher in animals exposed to transport stress. In the same species immunostaining to HSP70 antibody was observed only in red skeletal muscle and epidermis of control animals.
In the second part of the work we investigated about the use of rodlet cells as valuable tools to assess stressing conditions in fish. We focused on the localization of RCs by light and electronmicroscopy in gills of sea bass subjected to different conditions overcrowding. In general, a significant increase in number of rodlet cells has been observed in all animals subjected to overcrowding stress. In gills of control group rare rodlet cells were detected at the level of both primary and secondary lamellae, whereas in stressed group clusters of rodlet cells have been found in the epithelium of primary and secondary lamellae, indicating that these cells are influenced by stocking density.

Abstract (italian)

Scopo di questo progetto di lavoro è stato valutare la possibilità di utilizzare le “heat shock protein” 70 (Hsp70) e le “rodlet cells” (RCs) come parametri idonei alla valutazione dello stato di stress nei pesci, in particolar modo in alcune specie ittiche comunemente allevate. Nella prima parte del lavoro è stata valutata l’espressione delle Hsp70 inducibili e la localizzazione della stessa proteina, sia nella forma inducibile che costitutiva, in larve e avannotti di spigola (Dicentrarchus labrax) sottoposti a stress termico. E’ stata valutata anche, in parallelo, l’espressione e localizzazione delle Hsp70 in individui di spigola triploidi (coetanei) allevati nelle stesse condizioni e sottoposti allo stesso tipo di shock termico. I risultati ottenuti tramite RT-PCR confermano, sia per i diploidi che per i triploidi, l’avvenuta espressione della forma inducibile dell’Hsp70 in conseguenza dello stress termico. La localizzazione immunoistochimica delle Hsc70 (costitutive) conferma, sia nei diploidi che nei triploidi, la presenza pressoché ubiquitaria della proteina già a partire dai primi stadi di sviluppo e senza alcuna apparente dipendenza dall’esposizione allo stress termico. I tessuti maggiormente interessati dalla presenza della proteina sono, in genere, quelli di natura epiteliale: cute, mucosa del faringe e del tubo digerente. Stranamente non è stata osservata alcuna positività alla forma inducibile delle Hsp70, nonostante i risultati della RT-PCR confermino la presenza del messaggero.
E’ stata, inoltre, analizzata l’espressione della forma inducibile delle Hsp70 in larve, giovanili e adulti di spigola sottoposti a stress da trasporto e parallelamente è stata localizzata la proteina Hsp70 nelle sue due forme, inducibile e costitutiva, negli stessi animali. L’espressione delle Hsp70, analizzato tramite RT-PCR, non rileva differenze qualitative nella produzione del messaggero tra gruppi di controllo e stressati in larve ed avannotti, differenze che, a livello qualitativo, risultano invece apprezzabili negli adulti. L’analisi quantitativa (Real-Time PCR), però, ha permesso di rilevare differenze tra controlli e stressati significative per i giovanili di 80 dph ed appena evidenti per le larve di 40 dph. Mentre, negli adulti, l’espressione del messaggero per le Hsp70 è generalmente più elevata negli stressati che nei controlli. Immunoreattività alle Hsc70 (costitutiva) è stata riscontrata in diversi tessuti appartenenti ad animali in diversi stadi di sviluppo, senza differenze di rilievo tra gli animali sottoposti a stress da trasporto e quelli del gruppo di controllo. Immunoreattività alla forma inducibile delle Hsp70 non è stata riscontrata in alcun tessuto di spigola negli stadi più precoci di sviluppo.
Un esperimento analogo sullo stress da trasporto è stato effettuato successivamente utilizzando adulti di carpa (Cyprinus carpio) e trota iridea (Oncorhynchus mykiss). In questo caso è stata studiata solo la localizzazione della proteina e non l’espressione del messaggero. Nella carpa immunopositività all'Hsc70 (costitutiva) è stata riscontrata solo a livello della muscolatura rossa, sia nei controlli che negli stressati. Negli individui stressati, invece, è stata per la prima volta localizzata la presenza di Hsp70 (inducibile) nell'epitelio dei tubuli renali, oltre che nella cute e nelle branchie. Nella trota i risultati si rivelano molto simili a quelli evidenziati nella spigola per quanto riguarda le Hsc70, infatti diversi tessuti, sia dei controlli che degli stressati, esibiscono una netta positività.
Nella seconda parte del lavoro è stata valutata la possibilità di utilizzare le RCs come indicatori nei confronti di stress molto comuni nell’allevamento, quali il sovraffollamento e il confinamento. Sono state utilizzate spigole adulte sottoposte a due diverse densità di confinamento (20 Kg/m3 e 80 Kg/ m3) e per due diversi intervalli temporali (2 ore e 24 ore); al termine dell’esperimento è stato valutato il numero di RCs presenti nelle branchie alle diverse condizioni di stress. E’ stata inoltre effettuata un’analisi ultrastrutturale delle RCs al microscopio elettronico a trasmissione. Dopo 2 ore di confinamento gli individui mantenuti ad una densità di 80 Kg/m3 hanno evidenziato un aumento significativo nel numero di RCs, presenti nelle branchie, rispetto agli individui confinati ad una densità inferiore (20 Kg/m3). Dopo 24 ore il numero di RCs è aumentato ulteriormente, per entrambe le densità, ma in misura significativamente maggiore negli individui mantenuti ad una densità più elevata. In conclusione, il significativo aumento nel numero di RCs osservato nelle branchie di D. labrax già dopo due ore di stress da sovraffollamento, indica che queste cellule sono influenzate dalla densità di allevamento. Inoltre, l’organizzazione in “cluster” a livello di lamelle primarie e secondarie, supporta l’ipotesi che attribuisce alle RCs un ruolo di difesa nei confronti di eventi stressanti. Le RCs in definitiva possono a pieno titolo essere considerate dei validi indicatori nello studio di alcune forme di stress nei pesci.

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EPrint type:Ph.D. thesis
Tutor:Radaelli , Giuseppe
Ph.D. course:Ciclo 21 > Scuole per il 21simo ciclo > SCIENZE VETERINARIE > SCIENZE BIOMEDICHE VETERINARIE E COMPARATE
Data di deposito della tesi:01 February 2009
Anno di Pubblicazione:31 January 2009
Key Words:stress, specie ittiche allevate, "Hsp70", "rodlet cells", "fish welfare".
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/02 Fisiologia veterinaria
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Sperimentali Veterinarie
Codice ID:1857
Depositato il:01 Feb 2009
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