43 research outputs found
DESICCATION TOLERANCE AND HEAT-SHOCK PROTEINS EXPRESSION IN ACTIVE AND DORMANT AMPHIBOLUS VOLUBILUS (EUTARDIGRADA)
No full textInvertebrates living in extreme habitats at high altitudes or latitudes, as those living in ephemeral habitats, must be able to survive environmental stresses such as desiccation and freezing. In this contest several important scientific issues are emerging. Organisms can have four responses to overcome environmental stresses: regulative, acclimation, developmental, and evolutionary responses. Regulative and acclimation responses are carried out by the organisms in short or very short time to maintain their internal conditions for appropriate optimal functioning. Both responses are reversible in such a way as to follow the environmental fluctuations. They are also measurable according to a space-time scale depending on the size and the length of the life cycles of the organisms. Considering tardigrades, their persistence in unpredictable habitats is due to two widespread adaptive strategies, which are regulative responses: ability to enter cryptobiosis (both anhydrobiosis and cryobiosis) and/or to enter cyst. Anhydrobiosis and encystment are certainly characterized by several molecular events only partly identified. Other than the disaccharide trehalose, several stress proteins seem to be further keys to understand anhydrobiotic mechanisms. In particular, heat shock proteins (Hsps) and their molecular partners, which play diverse roles, including that of molecular chaperons, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion and degradation of other proteins.A moss-dwelling eutardigrade collected in Northern Apennines (Modena, Italy; 1700 m a.s.l.), Amphibolus volubilis, has been utilized in our lab to evaluate the survival strategies in unpredictable habitats. It is known that this species is able to enter anhydrobiosis and cryobiosis and to form cysts. Lab experiments on desiccation tolerance have been carried out considering three different values of air relative humidity (RH). Survival resulted directly related to the RH values, and high survival (about 80%) was obtained only with the highest RH value tested (85%). Desiccated animals were utilized to evaluate the Hsp70 and Hsp90 expression by means of SDS-Page and Western blotting analysis. Hsp expression was also evaluated in active animals and (only Hsp70) in encysted (type 2 cyst) animals. Quantitative comparisons of protein expression have been made among these three conditions. The level of both Hsp70 and Hsp90 is higher in desiccated animals than in active ones. Encysted animals have a Hsp70 level lower than both active and desiccated animals. These results indicates that Hsp induction due to desiccation is necessary to preserve cells from desiccation damages. Instead, the lower Hsp level found in cysts with respect to active animals could be explained hypothesizing a little need of these proteins in animals already protected from environmental stresses by strong cuticular layers
Thermotolerance and thermal acclimation in active tardigrades.
No full textThe ability of desiccated (anhydrobiotic) tardigrades to survive and to resist high temperature stresses (up to 100°C) is well-known, while the tolerance of active (hydrated) tardigrades to heat-shocks is still very little known. In order to increase our knowledge on thermal adaptations of active tardigrades, we have carried out lab experiments on three eutardigrade species to evaluate the ability to survive to heat-shock (considering lethal temperature - LT50 - and critical thermal maximum - CTmax), and the possibility of thermotolerance induction. We considered two terrestrial species, the moss-dwelling Amphibolus volubilis and the lichen-dwelling Ramazzottius oberhaeuseri, and one limnic species, Borealibius zetlandicus. These species differ each other in anhydrobiotic and cryobiotic abilities, substrate colonized.Starting from a uniform condition of tardigrades maintained for 24 h in water at 16°C, groups of hydrated tardigrades have been exposed for 1 hour to a heat-shock (different experiments from 26°C to 42°C). The presence of active animals (body movements) was evaluated immediately after heat-shock (t0), after 1h (t1) and after 24 h (t24) from heat-shock. Survival was represented by active animals at t24. For A. volubilis and R. oberhaeuseri the presence of active animals and survival was evaluated also after acclimation of 1 h (the first species at 28°C, the latter at 30°C) and subsequent heat-shocking to temperatures higher than 33°C. All species look thermotolerant, even though their survival significantly decreases with the increase of the stress temperatures. Both CTmax and LT50 are species-specific. The first one is 39.0°C for A. volubilis and 37.0°C for R. oberhaeuseri and B. zetlandicus. The latter is 35.1°C for A. volubilis, 33.6°C for R. oberhaeuseri and 33.0° for B. zetlandicus. The percentage of active animals changes according to the shock temperature and differs among the species. The number of animals with active movements often significantly increases between t0 and t1 for some temperatures, apart R. oberhaeuseri which shows a decrease at 28°C. There are not significant differences between t1 and t24 in the number of active animals, with the exception of an increase in A. volubilis at 30°C and 33°C. Both in A. volubilis and R. oberhaeuseri the acclimation produces significant survival increases of 10-40% for heat-shock temperature between 33°C–39°C, while at 40°C and 42°C there is not survival. These results demonstrate that tardigrades, even when active, have evident ability to survive heath stresses, even though sampled in different habitats and characterized by quite different cryptobiotic performances
Thermal tolerance and expression of Hsp70 in larvae of Diamesa spp.
No full textAir temperature and precipitation patterns are likely to change under a scenario of global climate warming, with consequent effects on water temperature and hydrology of running waters, especially of those fed by glaciers. Invertebrates living in these habitats (mainly Chironomidae Diamesinae) are extremely specialised and have developed unique survival strategies to face environmental constraints. The ability to survive heat-shocks was investigated in cold stenothermal species, threatened of extinction by global warming. IV instar larvae of Diamesa cinerella gr. were collected seasonally (from March 2005 to March 2006) in the Noce river (Trentino, NE Italy) at two altitudes (1300 and 2600 m a.s.l.). Larvae were acclimated in a thermostatic chamber for 24 h at 4°C and then exposed for 1 h to heat-shocks from 26 to 35°C. Survival was recorded 1 h after the shock and thermal tolerance was evaluated as LT50. D. cinerella gr. resulted thermotolerant (LT50 varied from 30.1 to 32.9°C). Hsp70 were detected and quantified both in heat-shocked and un-shocked larvae of D. cinerella gr and in un-shocked larvae of other Diamesa species by means of SDS-Page and immunoblotting. An increase of Hsp70 expression was detected in shocked larvae of D. cinerella gr. collected in summer. Constitutive stress proteins were found in all species maintained at 4°C. This could explain the high resistance to brief heat-shocks found in D. cinerella gr
Cryobiosis survival in tardigrades.
No full textEight species of eutardigrades collected in different terrestrial and freshwater habitats and characterized by different anhydrobiotic capabilities have been submitted to freezing and thawing experiments. The aim was to test: i. the interspecific capability to survive cryobiosis and the differences in recovery time to active life; ii. the relationship between survival and cooling rates. Starting from a uniform condition of tardigrades maintained for 24 h in water at 14°C, the cooling was performed according to three different protocols. Experiment 1: replicates for each species considered were put in 4 ml of water, frozen at three temperatures (-9°C, -20°C and -80°C) and kept frozen for six days. Experiment 2: replicates of Ramazzottius oberhaeuseri and Amphibolus volubilis were put in 4 ml of water, frozen in liquid nitrogen for two minutes and then stored at –9°C. Experiment 3: replicates of R. oberhaeuseri were placed in different amounts of water (2 ml, 1 ml and 0.5 ml), frozen at three different temperatures as in experiment 1 and kept frozen for six days. Before thawing, all frozen animals were firstly put or maintained at –9°C and then thawed at 14°C. Animals were examined both after 2.5 h and 24 h, considering alive the animals with evident and coordinated body movements. In experiment 1, R. oberhaeuseri, A. volubilis, Macrobiotus areolatus and Macrobiotus richtersi show a high survival at any tested temperature. Hypsibius dujardini, Borealibius zetlandicus and Diphascon cf. scoticum show a lower survival and differences in survival among each temperature. Survival of Dactylobiotus parthenogeneticus is null at any tested temperature. In most cases, the recovery time increases with the fall of the temperature and differs among the species. The differences among the species seem clearly related to the habitat and above all to the capability to carry out anhydrobiosis, suggesting a strong relationship between anhydrobiosis and cryobiosis. In experiment 2, all specimens of both species did not survive to freezing at –196°C. In experiment 3, R. oberhaeuseri shows high survival also when the animals are frozen in less than 4 ml of water, even though a survival decrease is recorded between –9°C and –80°C for all tested water amounts. In general, the survival is lower when the water freezing time is shorter (corresponding to a higher cooling rate). Moreover, a negative relationship between the water freezing time and the recovery time has been found: the shorter the water freezing time, the longer the recovery time. A too short water freezing time does not allow survival. These results can be easily explained by the need to produce a sufficient amount of protectants, or not enduring too many damages
Tardigrades in extreme habitats: morphological and molecular aspects in anhydrobiotic and encysted Amphibolus volubilis.
No full textIn terrestrial ecosystems tardigrades often inhabit transient systems that can support life only for a fraction of the year (e.g. arctic tundra, deserts, temporary ponds, mosses, lichens and leaf litter). Persistence of tardigrades in these harsh habitats is due to their ability to enter dormant states such as cryptobiosis and/or encystment. Cryptobiosis is directly induced by exogenous stimuli (desiccation: anhydrobiosis; freezing: cryobiosis) and it is promptly broken when the adverse conditions are removed. Encystment is under endogenous control and could be only indirectly induced by environment stimuli. Breaking encystment requires a specific cue that may or may not correspond to favourable environmental conditions.Amphibolus volubilis is a moss-dwelling tardigrade with a boreo-alpine distribution. Therefore, it represents a good model species to study the survival strategies in unpredictable environments. We verified that it is able both to enter cryptobiosis and to form cysts. Morphological changes of the body have been evidenced in anhydrobiotic tuns and cysts collected in nature from moss collected in a post-glacial valley (Northern Apennines, Modena, Italy, 1700 m a.s.l.) and/or induced in lab. Entering anhydrobiosis specimens contract longitudinally turning their body into a tun, lose most of their free and bound water and reduce or suspend their metabolism. The cyst is the result of a series of successive and continued morphological changes that are more complex than those involved in tun formation. Encystment involves repeated de novo synthesis of new cuticular structures (several cuticles with different ultrastructure, modified and unmodified buccal-pharyngeal apparatuses and claws) and a reduction of metabolism and loss of water lower than those involved in the anhydrobiotic state. The expression of Heat shock proteins (Hsps) have also been investigated in A. volubilis. Hsp 70 has been detected in active, anhydrobiotic and encysted specimens, evidencing that the synthesis of Hsp 70 in this species is not only related to desiccation stress. The synthesis of Hsp 70 was evidenced only in the anhydrobiotic state of the tardigrade Richtersius coronifer and related to tun formation (Ramløv and Westh, 2001)
Cysts of Amphibolus volubilis (Eutardigrada, Eohypsibiidae): seasonal dynamics and laboratory induction.
No full textIt is known that specimens of Amphibolus can carry out both cryptobiosis (anhydrobiosis and cryobiosis) and encystment and that they produce two types of cysts (type 1 and type 2). In Amphibolus nebulosus from Greenland, the type 1 cyst is related to winter and the type 2 to summer. In Amphibolus volubilis, found in Apennines (Monte Rondinaio, Italy), a detailed description of the encystment processes has been done, but not of the relationships between climatic factors and dynamics of the cyst 1 and cyst 2. Therefore, a study on seasonal dynamics of the A. volubilis cysts has been carried out with monthly samplings, from March 2003 up to March 2005. For each sampling, all specimens present in five-six replicates of 0.5 g of moss have been collected and analyzed.In all samplings, non-encysted and encysted animals (type 1 or type 2 cysts) have been found. The cyst trends are similar in the two considered years. Type 1 cysts have been found from November to March-April, but they were present in a very low percentage with respect to the total animals. Type 2 cysts have been found from June to October and are often more abundant than the non-encysted tardigrades. The two types of cysts never overlap. Correlation tests have evidenced that dynamics of type 2 cyst is positively related to the air temperature (T) and negatively related to the relative humidity (RH) of the air. The trend of type 1 cyst is negatively related to T, while no relationship to RH has been evidenced. The presence of non-encysted animals is negatively related to T and positively related to RH. Experiments of encystment induction have been performed in laboratory. In the same experimental conditions (same temperature, photoperiod, food and oxygen availability), non-encysted animals collected in April form type 2 cysts, whereas animals collected in November form type 1 cysts. The number of days to enter each type of cyst is related to experimental temperature. At the same temperature, the animals spent less time to enter in type 1 cyst than in type 2 cyst. In lab, the animals come out from type 2 cyst, if do not die, soon or later encyst again and enter always type 1 cyst.The data from nature and those from laboratory lead to conclude that, differently from what happens in anhydrobiosis and cryobiosis, climatic conditions do not directly affect encystment; but seasonal changing induces still unknown endogenous factors responsible of that kind of dormant stage
Risposte in modelli animali agli effetti dell’ambiente spaziale: i tardigradi, organismi resistenti a stress di ambienti estremi.
No full textI tardigradi rappresentano un modello animale particolarmente valido per la comprensione dei meccanismi, a livello organismico e cellulare, di protezione dagli stress dell’ambiente spaziale. Sono infatti invertebrati microscopici (circa 0,3-0,5 mm) che per condurre vita attiva necessitano di almeno un velo d’acqua, ma quando questa non è disponibile, rallentano notevolmente, o sospendono, il metabolismo sopravvivendo essiccati (anidrobiosi), o congelati (criobiosi). Entrando in anidrobiosi producono bioprotettori (es. trealosio) che stabilizzano le membrane cellulari. In anidrobiosi resistono inoltre ad ulteriori stress, come ad esempio, temperature di 150°C, o vicine allo zero assoluto (-273°C), radiazioni ionizzanti 500 volte superiori a quelle che porterebbero all’immediata morte dell’uomo, atmosfere sature di CO2 e HNO3, immersione in alcoli e pressioni di 600 Mpa (Bertolani et al., 2004). I tardigradi attuano la criptobiosi (anidrobiosi e criobiosi) in qualunque fase del ciclo vitale; alcune specie possono essere allevate (Altiero & Rebecchi, 2001) risultando particolarmente longeve, tanto da superare l’anno di vita (Altiero, Rebecchi & Bertolani, 2006). Inoltre, a differenza di altri minuscoli metazoi, sono caratterizzati da divisioni cellulari anche nell’adulto. Indagini dell’U.O. sulle capacità criptobiotiche dei tardigradi (parzialmente finanziate da ASI, 2001) hanno consentito di dimostrare la presenza di differenze intra- ed interspecifiche nella sopravvivenza in anidrobiosi (Jönsson, Borsari & Rebecchi, 2001), la relazione fra tratti fenotipici dei tardigradi e sopravvivenza all’essiccamento (Jönsson & Rebecchi, 2002), la sopravvivenza a lungo termine in anidrobiosi (Rebecchi et al., 2006), la presenza di differenze interspecifiche nella capacità di resistere al congelamento (Guidetti et al., inviato).L’ambiente spaziale provoca sui viventi stress che accelerano il tasso di invecchiamento; per individuare strategie contro l’invecchiamento ed anche al fine di applicazioni biotecnologiche, viene qui proposto lo studio dei meccanismi molecolari che consentono ai tardigradi di resistere agli stress tipici di tale ambiente, in modo da acquisire conoscenze sui meccanismi di riparazione e mantenimento di cellule e organismi. In questa prospettiva si colloca dunque il progetto ASSC-TARSE (TArdigrade Resistance to Space Effect), inserito nell’area II (Cellule, Tessuti, Organi) del progetto MoMa. Il progetto ASSC-TARSE, utilizzando tardigradi in toto e loro cellule (storage cells) in coltura primaria, si propone di: i. valutare la sopravvivenza dei tardigradi dopo stress quali essiccamento, microgravità e radiazioni attraverso il confronto fra animali attivi e anidrobionti; ii. acquisire informazioni sulla fisiologia, la biochimica e il genoma dei tardigradi a seguito di tali stress; iii. acquisire informazioni sull’apoptosi. Durante l’elaborazione del progetto, l’U.O. ha messo a punto i protocolli per l’allestimento di colture primarie di storage cells, l’analisi del DNA genomico e l’analisi dell’espressione di Heat-shock proteins (Hsp) nei tardigradi attivi ed anidrobionti. La disponibilità di tali protocolli, oltre all’ottenuta dimostrazione che i tardigradi essiccati esprimono più Hsp70 rispetto a quelli attivi, rappresentano una valida base di partenza per il raggiungimento degli obiettivi sopraindicati. La piena realizzazione del progetto TARSE consentirà di ottenere i seguenti risultati: i. la rilevazione degli effetti dell’essiccamento, delle radiazioni e della microgravità sulla sopravvivenza sia di organismi in toto che di cellule in coltura; ii. la rilevazione di probabili danni al DNA genomico e dell’eventuale capacità di riparo; iii. la rilevazione dei fenomeni di apoptosi; iv. l’individuazione e la quantificazione di bioprotettori coinvolti nei meccanismi di resistenza all’essiccamento e alle radiazioni: Heat-shock proteins (Hsp70 e Hsp90) e Late Embryogenesis Abundant proteins (LEA); v. lo studio del ruolo di queste biomolecole nei meccanismi di protezione, nell’ipotesi di un loro utilizzo come contromisure degli effetti dannosi correlati all’invecchiamento, o per sviluppare tecniche di conservazione a lungo termine di cellule, o tessuti
Expression of the 70 kDa Heat shock protein family in Alpine freshwater chironomids (Diptera, Chironomidae) under natural conditions
No full textChironomidae represent up to 100% of the fauna of Alpine streams. Because they survive stress conditions such as extremely lowtemperature (annual mean cold and expression of stress proteins such as the 70 kDa Heat shock protein family. Fourth instar larvae of ten species of coldstenothermalchironomids (Pseudodiamesa branickii, Diamesa latitarsis, D. laticauda, D. cinerella, D. insignipes, D. zernyi, D.vaillanti, Orthocladius (O.) frigidus, O. (Euorthocladius) thienemanni and Paratrichocladius nivalis) were collected in a glacier-fedstream in NE Italy at two stations (1300 and 2600 m a.s.l.) and in two seasons (summer 2005 and spring 2006). Immunodetectionand quantification of the relative levels of Hsp70 family were performed via Western blot analysis. Significantly different levels ofHsp70 were detected among species. The highest amounts were recorded in P. nivalis and D. insignipes, the lowest in P. branickii.Within the genus Diamesa, lower levels of Hsp70 were observed in the most cold-stenothermal species than in the less coldstenothermalones. These differences may be explained by different species autoecology. The results provide information onbiochemical strategies of alpine midges to face cold temperatures under natural conditions and new insights into their possibleresponse to global warming
Heat shock proteins in encysted and anhydrobiotic eutardigrades
No full textHeat shock proteins (Hsps) can help organisms to survive environmental stresses. Tardigrades are aquatic metazoans able to colonize unpredictable, or “hostile to life”, terrestrial habitats entering resting stages such as cysts and anhydrobiotic tuns. In this paper we compared the Hsp70 and Hsp90 expression between resting stages (tuns or cysts) and active hydrated specimens of two eutardigrade species, namely Bertolanius volubilis and Ramazzottius oberhaeuseri. The two species partly differ in the kind of dormant stages utilized and in habitats colonized. In both species desiccation stress did not induce an up-regulation of either Hsps. Our data, together with those from literature, suggest that in tardigrades Hsps are involved in repairing molecular damages after anhydrobiosis, rather than in the stabilization of molecules during the dry state. Finally, the first demonstration of the presence of Hsps in diapausing cysts of B. volubilis are reported and discussed
Caterina Tarabotti Unveiled
No full textCaterina Tarabotti (1615–1693) never identified herself as a painter in contemporary
documents or claimed authorship of any picture with her signature. She would have
surely joined the ranks of the many anonymous women painters of her time, were
it not for the art critic and historian Marco Boschini (1602–1681). In his La Carta
del Navegar Pitoresco (1660), Boschini was the first to notice Caterina’s talents and
provide information about her life.1
Alluding to her namesake saint and surname,
Boschini infused the painter’s biography with lyric flair. A light coming out of the
Varotari workshop, Caterina was, according to Boschini, a virgin very gifted in
painting. She herself honored art with her achievements, which came effortlessly.
Comparing her to a silver lamp that never burns out, the author, with a sense of
optimism, concluded that Caterina would live forever and her name would bear
eternal light and splendor
