Alternative matrices for cortisol measurement in fish.

Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish in which blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol significantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing different cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish

Heat Shock Protein 70 e Rodlet Cells come possibili indicatori di stress in specie ittiche comunemente allevate

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.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

Effetti dello stress da trasporto sulla produzione di cortisolo e HSP70 in spigole (Dicentrarchus labrax) a diversi stadi di sviluppo.

Stress conditions due to transport procedure were highlighted in Dicentrarchus labrax (Osteichthyes, Perciformes) larvae, post larvae and adults. As stress indicators, variation in cortisol levels in whole body or plasma, muscle, mucus and faeces were detected. Also the expression of constitutive and inducible heat shock protein 70 (HSP70) was evaluated to determine the cellular response to stress

Supporting the Development of Next-generation Fog Services

Fog Computing is a recent and compelling paradigm that proposes to run information-processing services at the edge of the network. While interesting standardization efforts are being currently pursued by many organizations, most of them focus on management and orchestration functions and primarily propose the adoption of programming models designed for Cloud applications in the Fog. Instead, Fog Computing applications would significantly benefit from innovative solutions that adopt an acceptable lossiness perspective and manage information processing and dissemination in a dynamic and integrated fashion. This paper presents an overview of the opportunities and challenges in Fog Computing application development, proposes an innovative holistic Software Defined Networking (SDN) approach based on an information-centric and value-based service model to support those applications, and presents an overview of the Holistic Analytics and Networking (HAN) SDN architecture that we are developing to realize this ambitious vision

Alternative indicators to evaluate stress in sea bass (Dicentrarchus labrax, L.)

Joint international conference & exhibition of European Aquaculture Society & World Aquaculture Societ

Gene and protein expression analysis of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax

This study reports the expression and cellular localization of insulin-like growth factor-I (IGF-I) and myostatin (MSTN) during the life cycle of sea bass (Dicentrarchus labrax) by RealTime PCR, in situ hybridization (ISH) and immunohistochemistry. RealTime PCR indicated that IGF-I mRNA increased significantly during the first week post-hatching (ph). In adults, the highest expression of IGF-I mRNA was observed in liver whereas MSTN mRNA was almost exclusively detected in muscle. ISH showed IGF-I mRNA expression in liver already at 8 days ph and, at 25 days ph, also in growing cartilage. MSTN mRNA increased significantly from day 25 and the expression of mRNA was higher in muscle and almost paltry in other organs/tissues. Interestingly, the lateral muscle showed a quantitative differential expression of IGF-I and MSTN mRNAs in red and white muscle, depending on the developmental stage examined. IGF-I immunoreactivity was detected at hatching in developing intestine, skeletal muscle, skin and yolk sac. At hatching, MSTN immunostaining was evident in skeletal muscle, skin and yolk sac. From the fourth day ph IGF-I and MSTN proteins were detected in liver and, subsequently, in kidney and heart muscle from day 10. These results suggest that IGF-I and MSTN are involved in the regulation of somatic growth in the sea bass

Valutazione, con Daphnia magna, della tossicità acuta di quattro antibatterici

Oxytetracycline (OTC), tylosin (TYL), sulfadimethoxine (SDM) and sulfamethazine (SMZ) are largely employed for mass treatments in food animals and so prone to contaminate the environment. According to the EMEA guidance document on Environmental Impact Assessment for veterinary drugs, a Predicted No Effect Concentration in water should be derived from the acute toxicity test in Daphnia magna. Using Daphtoxkit® (compliant with OECD Guideline 202) their toxicity on Daphnia magna was evaluated. Results shows a low sensitivity to OTC and SDM, confirms a moderate sensitivity to TYL (EC50=1012 mg/L) and highlights the particular sensitivity to SMZ (EC50=112 mg/L). This is of interest because SMZ is one of the most widely used sulfonamide and has been detected in soil and in wells

Use of sulfadimethoxine in intensive calf farming: evaluation of transfer to stable manure and soil

After prophylactic treatment of 50 calves with 62 mg kg-1 day-1 of sulfadimethoxine (SDM) for five days, the levels of the drug over time were followed in feces, bedding and stable manure, and then in the soil of a manured field and surrounding drainage courses. Analysis were done by HPLC after applying to the different matrices a quick and simple extraction procedure. The half-life of the drug in bedding was very short (24 h). In stable manure the degradation rate of the drug slowed down and the calculated half-life was 64 days, with 390 ug kg-1 of SDM still detectable after three months maturation. However, in a five months matured stable manure obtained from other groups of calves subjected to the same prophylactic treatment, levels of SDM were <50 lg kg-1 (LOD of the analytical method). After field fertilization with this manure, no traces of SDM were found in soil (LOD 10 ug kg-1) or in the water (LOD 2 ug l-1) from the surrounding drainage courses. Using the internationally recognised DAPHTOXKIT-FTM, a SDM toxicity test toward Daphnia magna was performed in the range 10\u2013100 mg l-1. The test gave negative results both after 24 and 48 h, confirming that microcrustaceans are less sensitive than other models to the toxicity of antibacterials. However, based on data from other authors, concerning algal toxicity and microbial inhibition, and on the analytical results from the current field study, the calculated worst-case EC50/PEC ratio for SDM both in freshwater and in soil was still >1000