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Anidrobiosi, radiazioni ultraviolette e riscaldamento globale: adattamenti ecologici, fisiologici, biochimici e molecolari nei tardigradi
No full textObiettivo del mio progetto di dottorato è stato l’identificazione di strategie adattative di tipo ecologico, fisiologico, biochimico e molecolare evolute dai viventi per tollerare l’essiccamento naturale e l’aumento di temperatura e radiazioni ultraviolette (UV) dovuti ad alterazioni della quantità di ozono troposferico e di CO2.
Come organismi modello sono state usate due specie di tardigradi (Acutuncus antarcticus, specie endemica dell’Antartide, e Paramacrobiotus richtersi, specie di zone temperate) di cui sono state confrontate le risposte adattative. I tardigradi sono animali acquatici microscopici in grado di colonizzare habitat terrestri, grazie alla loro capacità di entrare in anidrobiosi, condizione fisiologica in cui la vita attiva è sospesa perché viene persa per evaporazione fino al 97% dell’acqua corporea. In anidrobiosi questi micrometazoi possono rimanere vitali per decenni ed essere in grado di tollerare stress fisici e chimici non tollerati da altri animali.
L’analisi biochimica svolta su esemplari di P. richtersi seccati sperimentalmente ha evidenziato un accumulo di specie reattive all’ossigeno (ROS) durante la permanenza in anidrobiosi. Di conseguenza, è stata valutata l’attività degli enzimi antiossidanti e la quantità di glutatione durante la cinetica dell’anidrobiosi in entrambe le specie. In P. richtersi, l’attività della catalasi aumenta significativamente con l’essiccamento e diminuisce con la reidratazione. In A. antarcticus, sono l’attività della superossido dismutasi e il contenuto di glutatione a diminuire significativamente negli animali secchi e ad aumentare in quelli reidratati. Mediante la tecnica molecolare “RNA interference” sono stati silenziati geni codificanti per molecole potenzialmente coinvolte nella tolleranza all’essiccamento (enzimi antiossidanti, proteine da shock termico, acquaporine e trealosio) di P. richtersi, per comprendere il loro ruolo nell’anidrobiosi. I risultati mostrano il coinvolgimento dell’enzima glutatione perossidasi come importante sistema di eliminazione di radicali liberi.
Per valutare la capacità dei tardigradi di far fronte agli stress correlati al riscaldamento globale, sono stati valutati la capacità di sopravvivenza e i tratti del ciclo vitale di A. antarcticus dopo essiccamento ed esposizione ad alte temperature e radiazioni UV. Il ciclo vitale di A. antarcticus è corto, con un output riproduttivo basso. Questi tratti vitali rappresentano una valida strategia adattativa per sfruttare appieno la breve estate antartica che offre condizioni ambientali favorevoli per crescita e riproduzione. Esemplari idratati di A. antarcticus sono in grado di tollerare alte temperature, e sia animali attivi che secchi mostrano una buona tolleranza alle radiazioni UV. Questa tolleranza è stata valutata anche irraggiando con diverse dosi di UV uova a diversi stadi di sviluppo e raccogliendo dati sui tratti del ciclo vitale di due generazioni successive. Le uova irradiate hanno mostrato un ritardo nel tempo di schiusa rispetto a quelle non irradiate. Inoltre, sono stati osservati effetti negativi sui tratti del ciclo vitale e difetti morfologici nella prole. Infine, la sopravvivenza di A. antarcticus diminuiva negli animali esposti simultaneamente alle radiazioni UV e a temperature crescenti. I risultati ottenuti su A. antarcticus sono stati confrontati con quelli presenti in letteratura o ottenuti in questa tesi (tolleranza alle alte temperature) per P. richtersi.
Il mio progetto di dottorato ha permesso l’identificazione di varie strategie adattative evolute dai tardigradi per tollerare condizioni ambientali sfavorevoli, e conferma la validità dei tardigradi come organismi modello per migliorare la conoscenza scientifica sui meccanismi naturali di difesa evoluti dagli animali.The aims of my research were the identification of ecological, physiological, biochemical, and molecular defense strategies naturally evolved by animals to tolerate complete desiccation, and increasing UV radiation and temperature due to the alteration of the tropospheric ozone budget and alteration of the CO2 amount.
To carry out my research, two species of tardigrades (Acutuncus antarcticus from Antarctic region, and Paramacrobiotus richtersi from temperate region) were used as model animals, and their responses to stress conditions were compared. Tardigrades are microscopic aquatic animals able to colonize terrestrial habitats thanks to their capability to enter anhydrobiosis, a temporary suspension of active life due to the loss up to 97% of body water. They can persist in anhydrobiosis for decades and in this physiological state are able to withstand several physical and chemical extreme stresses.
Biochemical analysis performed in experimentally desiccated specimens of P. richtersi demonstrated the accumulation of Reactive Oxygen Species (ROS) during the permanence in the anhydrobiotic state. As a consequence, the activities or accumulation of antioxidant molecules (e.g. scavenging enzymes and glutathione) during the kinetic of anhydrobiosis was evaluated in both model species. In P. richtersi, the catalase activity increased significantly during desiccation process and decreased during rehydration. In A. antarcticus, the activity of superoxide dismutase and the content of glutathione significantly decreased in desiccated animals and increased during rehydration. RNA interference (RNAi) molecular technique was utilized to silence genes enconding to molecules potentially involved in anhydrobiosis (e.g. scavenging enzymes, heat shock proteins, aquaporines, trehalose) in order to fully understand their role in desiccation tolerance of P. richtersi. The results point to the involvement in the desiccation process of at least glutathione peroxidase, a very important antioxidant system in scavenging free radicals. .
To evaluate the capability of tardigrades to cope with stress related to global warming, the survival and life history traits of A. antarcticus were analysed on animals and eggs after desiccation and exposition to increasing temperature and UV radiation. The life cycle of A. antarcticus is short and its reproductive output was low, with a short generation time. These traits are advantageous for exploiting the conditions suitable for growth and reproduction during the short Antarctic summer. Hydrated animals of A. antarcticus were able to tolerate increasing temperature values, and both hydrated and desiccated animals showed a good tolerance to increasing UV radiation doses. This tolerance was evaluated also irradiating eggs at different developmental stage and collecting life history traits of two successive generations. All irradiated eggs showed a delay in the hatching time with respect to not irradiated eggs. Moreover, a negative effect on life history traits and newborns with morphological defects were observed. Furthermore, the survivorship of A. antarcticus animals decreased when they were simultaneously exposed to UV radiation and increasing temperatures. These new data on A. antarcticus were compared with those existing and newly produced (tolerance to high temperature) on the temperate species P. richtersi.
The data obtained during the PhD project allowed to identify the strategies evolved in tardigrades to withstand stressful environmental conditions, and confirmed tardigrades as a reliable animal model system to improve scientific knowledge on natural defense mechanisms evolved in animals
FIGURE 3. A in An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens
No full textFIGURE 3. A: Egg shell of M. terminalis (hologenophore C2868-N02 US2, DIC); B: Egg shell of M. terminalis by SEM (C2868); C: Egg shell of a M. terminalis paratype (C624-S44, PhC); D: Egg shell of M. cf. terminalis (C2341, PhC). Bar =10 µm (A, C, D); 1 µm (B).Published as part of Cesari, Michele, Giovannini, Ilaria, Bertolani, Roberto & Rebecchi, Lorena, 2011, An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens, pp. 42-51 in Zootaxa 3104 (1) on page 48, DOI: 10.11646/zootaxa.3104.1.3, http://zenodo.org/record/524530
FIGURE 1. A in An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens
No full textFIGURE 1. A: Fresh specimen of M. terminalis; buccal pharyngeal apparatus with dorsal buccal armature (C2868, DIC); B: Fresh specimen of M. macrocalix; buccal pharyngeal apparatus with dorsal buccal armature (C2868, DIC). C: Claws with indented lunules on a hind leg of M. terminalis (C2868, SEM); D: Claws with indented lunules (arrow heads) on the hind legs of a fresh specimen of M. terminalis (C2868, DIC). Bar =10 µm (A, B, D); 5 µm (C)Published as part of Cesari, Michele, Giovannini, Ilaria, Bertolani, Roberto & Rebecchi, Lorena, 2011, An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens, pp. 42-51 in Zootaxa 3104 (1) on page 46, DOI: 10.11646/zootaxa.3104.1.3, http://zenodo.org/record/524530
Desiccation tolerance and production of Reactive Oxygen Species (ROS) in the anhydrobiotic water bear Paramacrobiotus richters
No full textThe production of Reactive Oxygen Species (ROS) during desiccation processes is documented in bacteria and plants, whereas studies on animals are in practice lacking. In this study we investigated the response to dehydration with respect to the ROS production during the kinetic of the desiccation process and the short and long-time permanence in the anhydrobiotic state. We hypothesized that ROS production, and thus oxidative damage, may be greater in animals maintained desiccated for long-time than in animals maintained dry just for one day. The tardigrade Paramacrobiotus richtersi was used as a target animal. In parallel, oxidative stress was assessed in hydrated P. richtersi as control. Adults were experimentally dehydrated in laboratory using an optimal desiccation protocol to achieve a 100% survival rate of P. richtersi. Animals were maintained desiccated (at 3% RH and 20°C) from 1 to 40 days. Intracellular ROS production during rehydration was evaluated in the “storage cells” (free-floating cells in the tardigrade body cavity), after treatment of the tardigrades with the probe 2,7-dichlorodihydrofluorescein-diacetate (DCFH2-DA). The amount of green fluorescent oxidation product (DCF), which reflects the reaction of the probe with intracellular free radicals, was measure by a laser scanner confocal microscope. The desiccation process does not produce a high amount of ROS, even though previous studies on the same species, P. richtersi, demonstrated an increase of antioxidant enzyme activity in desiccated specimens with respect to hydrated ones. Instead, the long-time permanence in anhydrobiosis (20 days) produces a significant increase of ROS, as evidenced after three and twelve hours from rehydration. The collection of data about the permanence in anhydrobiosis for 40 days is in progress. The high amount of ROS detected in animals maintained dry for long-time, and consequently the oxidative molecular damages, justifies that tardigrade survival decreases with the time spent in a dry state
Essiccamento e produzione di specie reattive dell’ossigeno (ROS) in tardigradi anidrobionti
No full textAnche se l’acqua è essenziale per la vita, organismi di linee evolutive diverse hanno sviluppato la capacità di tollerare un essiccamento estremo entrando in un particolare stato fisiologico reversibile, detto anidrobiosi. Con questo adattamento, rotiferi bdelloidei, nematodi e tardigradi perdono fino al 97% dell’acqua corporea, con sospensione del metabolismo e cambiamenti nell’organizzazione e composizione molecolare delle membrane cellulari. Nei tardigradi essiccati, la sopravvivenza a lungo termine è inversamente proporzionale a temperatura, umidità relativa dell’aria e pressione parziale di ossigeno, fattori abiotici che contribuiscono a danneggiare le molecole biologiche. Una delle principali cause di danno durante l’anidrobiosi sembra essere lo stress ossidativo, dovuto al disequilibrio fra l’eccessiva produzione di specie reattive dell’ossigeno (ROS) e la limitata attività degli antiossidanti. Tuttavia, la produzione di ROS durante l’essiccamento è ben documentata solo in pochi organismi, soprattutto autotrofi, mentre mancano praticamente dati sugli animali anidrobionti e, soprattutto, sull’effettivo accumulo di ROS durante la permanenza in anidrobiosi per lunghi periodi di tempo.
È stata quindi valutata la produzione di ROS nell’eutardigrado anidrobionte Paramacrobiotus richtersi, analizzando animali essiccati sperimentalmente in laboratorio [4 h a 18°C e 80% di umidità relativa dell’aria (RH); 4 h a 18°C e 50% RH; 12 h in gel di silice] e mantenuti essiccati per vari periodi di tempo (da 1 a 30 giorni) o sottoposti a temperature elevate (37°C e 60°C). Come controllo sono stati utilizzati animali mantenuti idratati. La produzione di ROS è stata valutata nei globuli cavitari, cellule libere nella cavità corporea dei tardigradi, dopo trattamento degli animali con il marcatore 2,7 diclorodiidrofluoresceina diacetato (DCFH2-DA) e successiva rilevazione della quantità del prodotto di ossidazione fluorescente 2,7 diclorofluoresceina (DFC), mediante microscopia confocale a scansione laser.
I primi dati indicano che in P. richtersi l’entrata in anidrobiosi di per sé non determina la produzione di elevate quantità di ROS, sebbene sia stato dimostrato un incremento dell’attività degli enzimi antiossidanti negli esemplari essiccati rispetto a quelli idratati. La produzione di ROS sembra aumentare quando gli animali essiccati sono mantenuti in anidrobiosi per lunghi periodi di tempo ed esposti ad alti valori di temperatura
An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens
No full textWe have in recent papers revealed that an integrative taxonomy approach helps to solve taxonomic problems in tardigrades. However, whole tardigrades are required for DNA work, which leaves no hologenophore voucher specimens with adult morphology. Using a novel methodology for the Tardigrada, we introduce the practice of collecting high quality maximum magnification light microscopy images of recently thawed animals to act as hologenophore voucher specimens of animals later used for DNA barcode sequencing. Within the framework of a DNA barcoding project on tardigrades, we collected a moss sample from the type locality of Macrobiotus terminalis Bertolani & Rebecchi, 1993 (Castelsantangelo, Central Apennines, Italy), a species of the “Macrobiotus hufelandi group”. Within the moss sample we found several animals and eggs with a morphology that corresponded to the original description of M. terminalis, while others were attributable to Macrobiotus macrocalix Bertolani & Rebecchi, 1993. In this study, molecular (cox1 mtDNA) analyses demonstrated no intraspecific variability in M. terminalis from the type locality but very large interspecific differences when compared with M. macrocalix and GenBank data for other species within the M. “hufelandi group”. There was also a large difference between our M. terminalis sequences and the GenBank data of a specimen attributed to the same species. The GenBank sequence originated from a population in the Northern Apennines, whose morphology appeared to be like that of the specimens of the locus typicus. This confirmed the importance in utilising material from the type locality for linking molecular data to the species’ morphological characters. Our paper underlines the importance of an integrative taxonomy in species diagnoses and demonstrates a scenario where morphological observations alone are not always sufficient. Lastly, this work adds reliable information to the sequence reference library that provides a useful building block for further studies on similar and related tardigrade taxa
DNA barcoding and integrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the first tardigrade species to be described, and some related species
No full textWithin the framework of a DNA barcoding project on tardigrade species, a study was carried out on Macrobiotus hufelandi C.A.S. Schultze 1834, the first formally described tardigrade species. We used samples collected from the type locality and additional material from other European sites containing species of the “M. hufelandi group”. The study was performed by integrating morphological, karyological and molecular (mt-DNA cox1) information and comparing these data with morphological data from the type material. Several species from this group were found in the type locality of M. hufelandi (near Freiburg, Black Forest, Germany) and these were all barcoded. One was M. hufelandi, the other two were: Macrobiotus sandrae Bertolani & Rebecchi 1993 (originally described from the same locality), and Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari in press (type locality Andalo, Italy), all with interspecific genetic distances of more than 19%. A fourth cryptic species, which had the same morphology as M. hufelandi but a genetic distance of 6.7%, was not described as a new taxon but named M. cf. hufelandi sp.1 for this study. Macrobiotus sandrae and M. vladimiri were also present (and barcoded) in Italy (Alps). Additional individuals (animals and eggs) were also found, and barcoded, in Italy (Apennines) and Switzerland that belonged to the haplogroup Macrobiotus cf. hufelandi sp. 1. These data together with other recent studies on tardigrade DNA barcoding represent a starting point for further studies on tardigrade biogeography, phylogeography and diversity
The toughest animals on the Earth: desiccation tolerance and oxidative stress in tardigrades.
No full textTardigrades are microscopic aquatic ecdysozoans with remarkable abilities to withstand harsh physical conditions, such as dehydration or exposure to harmful highly-energetic radiations, including both solar and cosmic ionizing radiations and the vacuum of the space. They can persist in dehydrated state even for years at any life stage. Yet once external conditions become favorable they resume an unaffected active life. Nevertheless, survival of desiccated tardigrades decreases with the dehydration rate and time spent dry. On the other hand, experimental studies on Paramacrobiotus richtersi provide evidence that exposure to high temperatures, high humidity, and high oxygen partial pressure negatively affect long-term survival of anhydrobiotic tardigrades, and directly influence the time required to reactivate their metabolism. These abiotic factors produce molecular damages, which are accumulated in proportion to the time spent in the desiccated state, potentially leading to tardigrade death. Oxidative stress seems to be one of the most deleterious causes of damages due to water depletion. Experimental studies on P. richtersi indicate that the production of Reactive Oxygen Species (ROS) could occur during anhydrobiosis, being the accumulation of ROS higher in tardigrades maintained desiccated for long-time than in those maintained desiccated for one day only. Therefore anhydrobiosis needs a stringent control of oxidation processes including ROS production. Experimental studies evidence that glutathione and ROS scavenging enzymes represent a key group of molecules for desiccation tolerance in P. richtersi, where the activity of these enzymes is significantly higher in desiccated specimens than in hydrated specimens. We also suggest a role of tardigrade pigments (e.g. carotenoids) as scavengers for ROS forming during dehydration processes and/or exposition to solar radiations.
These data exhibit further evidence on the role of antioxidant defenses in tolerant desiccation organisms, and the role of tardigrades as animal model to discover the secret of life without water
The short life cycle of Acutuncus antarcticus (Tardigrada) as adaptation to Antarctic environment
No full textThe climate global change is altering the tropospheric ozone budget increasing harmful ultraviolet (UV) radiation with consequences on human and ecosystem health. Antarctic micrometazoans are particularly vulnerable to the synergic effects of increasing temperature and UV radiation as their growing season matches with the spring period of ozone depletion. The roles that phenotypic plasticity and genotypic evolution play in whether Antarctic organisms can cope with these environmental changes are not fully understood. Tardigrades represent one of the main terrestrial components of Antarctica fauna in terms of distribution, number of specimens and colonized substrates. Studies on adults of the eutardigrade Acutuncus antarcticus, one of the most abundant species in Antarctic bryophytes and freshwater sediments, showed that they survived to experimental exposition to increased temperature and UV radiation. These results suggest that A. antarcticus could survive to possible environmental changes. To test this hypothesis we firstly needed to know its life history traits, never studied before, in order to carry out further experiments on stress resistance of eggs and juveniles.
Cultures were started using single specimens of A. antarcticus collected from a temporary freshwater pond at the Italian Antarctic base at Victoria Land. Animals were fed with the algae Chlorococcus sp., and reared at 14°C and 12h/12h L/D.
A. antarcticus lays freely (rarely within exuvium) eggs hatching in 7-9 days. Newborns molt 2-3 times before their first oviposition that occurs at the age of 12-15 days. Successive ovipositions occur once a week (for 2-3 months) and are always preceded from a molting. The number of laid eggs (1-4 per oviposition) is a function of the female age. Throughout lifespan (3-4 months) each female lays up to 20 eggs. Individually reared newborns reached sexual maturity and, maintained isolated, laid eggs able to hatch. Males were never seen. Therefore this population reproduces via thelytokous parthenogenesis.
The life cycle evidenced in A. antarcticus, characterized by a short generation time with respects to that of species of temperate regions, represents an adaptive strategy which allows animals to be active and reproduce only during the short-time (ca. 2 months) in which water is available. This knowledge will allow further experiments to verify the eventual effects of temperature and UV radiation on the life history traits and fitness of Antarctic organisms
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