Assessing the expression of HsP60 in scleractinian corals subjected to biotic and abiotic stresses

The reef health worldwide is seriously threatened by a multitude of factors such as abnormally elevated and low ocean temperatures, high UV radiations, changes in salinity, pollution and increasing incidence of diseases. Under adverse circumstances the equilibrium between the partners of the coral holobiont may be compromised and can lead to coral bleaching events. Bleaching refers to the loss in the coloration of the coral colony induced by the dissociation of the symbiosis between corals and their symbiotic algae. The extreme or unexpected environmental fluctuations could be very stressful for sessile marine organisms such as corals, causing important cell damage since corals lack any developed physiological regulatory system. One mechanism of reaction to deleterious conditions is the rapid increase of the induction of a set of stress proteins called Heat shock proteins (Hsps). Under normal cellular physiological conditions the Hsps mainly function as molecular chaperones and they are involved in a multitude of proteome-maintenance functions that regulate protein homeostasis in directing the folding and assembly of other proteins. They also are involved in the intracellular protein transport and in the degradation of damaged proteins. An up-regulation of the expression of Hsps constitutes an emergency response and confers tolerance to harsh conditions. This study highlights the modulation of the expression of a vital but scarcely investigated group of Hsps, the mitochondrial Hsp60 which are essential for the vitality of the cell and whose induction represent one of the first reaction to stress. The overall objective of my dissertation is to elucidate the major aspects of Hsp60 modulation in various taxa of corals as a result of their exposure to different abiotic and biotic stress factors. In the first study we investigated for the first time the effectiveness of the Hsp60 as indicator of biotic stress and competitive interaction in the coral Acropora muricata, focusing on two biological interactions such as a coral disease, the Skeleton eroding band (SEB) caused by the protozoan Halofolliculina corallasia and the algal overgrowth. The two different biological stresses trigger diverse responses on Hsp60 level. No detectable effect on Hsp60 modulation appeared in colonies subjected to algal overgrowth. On the contrary, corals displayed a robust up-regulation of Hsp60 in the fragments sampled just above the SEB dark band indicating that the aggressive behavior of the protozoan causes cellular damage also in coral portions neighboring and along the advancing front of the infection. Portions of coral sampled distant to the SEB band showed a Hsp60 level comparable to that observed in healthy colonies. We propose Hsp60 expression as a promising tool to evaluate physiological stress caused by coral disease in reef corals. In the second study we examined the different modulation of Hsp60 in the coral Seriatopora caliendrum subjected to salinity stress, since that corals are generally considered stenohaline and osmoconformers. We analyzed the Hsp60 expression profiles of the coral polyps under three salinity scenarios (hypersalinity of 45 ppt, hyposalinity of 27 ppt and extreme hyposalinity of 15 ppt) during the time course of a 2 days period. Experiments were conducted at the Civic Aquarium of Milan using a flow-through aquaria system. S. caliendrum responds differently to hyper- and hyposaline conditions at morphological and cellular levels and the response of corals to osmotic stress reflects the severity and duration of the disturbance. The Western blot analysis showed for each salinity a similar strong up-regulation of Hsp60 after the first 6 h of stress, but subsequently Hsp60 exhibited for each salinity treatment specific patterns of expression. In hypersalinity condition a negative trend of Hsp60 expression was observed, but the colonies showed a morphological appearance similar to healthy control colonies, suggesting a possible metabolic acclimation of corals to the stress. In S. caliendrum exposed to moderate hyposalinity, Hsp60 exhibited marked oscillation and the level of Hsp60 generally remained high over time indicating that cellular damages in the animal host were in progress. In extreme hyposalinity condition, a considerable gradual down-regulation of Hsp60 was detected until the end of the experiment. This was accompanied by extreme degradation and necrosis of coral tissues. Finally, we focused on the responses of Hsp60 to thermal stresses, initially analyzing the susceptibility of three coral genera (Montipora, Acropora and Seriatopora) to a severe heat stress of 36°C for 12 h. Despite the Hsp60 trend appeared similar, each genus displayed a different persistence of the Hsp60 signal, and so a different threshold of tolerance and resistance. Secondly, the sensitive S. caliendrum was subjected to a cold shock of 21°C, a moderate heat shock of 29°C and a severe heat shock of 34°C. The modulation of the Hsp60 at lowered temperatures are similar to those involved in very elevated temperature stress with an up-regulation after 6 h followed by a down-regulation when the cellular damage become irreparable. This is accompanied by the appearance of bleaching events. The mild heat shock of 29°C did not significantly affect the normal Hsp60 oscillatory pattern. With this study we proposed the application of the mitochondrial Hsp60 and the analysis of its modulation as an useful and accurate biomarker, to assess the effect of several types of stress in scleractinian corals, and to diagnose coral health prior that the coral bleaching occurred.