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2022 - Antioxidant Response during the Kinetics of Anhydrobiosis in Two Eutardigrade Species [Articolo su rivista]
Giovannini, Ilaria; Corsetto, Paola Antonia; Altiero, Tiziana; Montorfano, Gigliola; Guidetti, Roberto; Rizzo, Angela Maria; Rebecchi, Lorena

Anhydrobiosis, a peculiar adaptive strategy existing in nature, is a reversible capability of organisms to tolerate a severe loss of their body water when their surrounding habitat is drying out. In the anhydrobiotic state, an organism lacks all dynamic features of living beings since an ongoing metabolism is absent. The depletion of water in the anhydrobiotic state increases the ionic concentration and the production of reactive oxygen species (ROS). An imbalance between the increased production of ROS and the limited action of antioxidant defences is a source of biomolecular damage and can lead to oxidative stress. The deleterious effects of oxidative stress were demonstrated in anhydrobiotic unicellular and multicellular organisms, which counteract the effects using efficient antioxidant machinery, mainly represented by ROS scavenger enzymes. To gain insights into the dynamics of antioxidant patterns during the kinetics of the anhydrobiosis of two tardigrade species, Paramacrobiotus spatialis and Acutuncus antarcticus, we investigated the activity of enzymatic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) and the amount of non-enzymatic antioxidants (glutathione) in the course of rehydration. In P. spatialis, the activity of catalase increases during dehydration and decreases during rehydration, whereas in A. antarcticus, the activity of superoxide dismutase decreases during desiccation and increases during rehydration. Genomic varieties, different habitats and geographical regions, different diets, and diverse evolutionary lineages may have led to the specialization of antioxidant strategies in the two species

2022 - Resistance to Extreme Stresses by a Newly Discovered Japanese Tardigrade Species, Macrobiotus kyoukenus (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Cesari, Michele; Giovannini, Ilaria; Altiero, Tiziana; Guidetti, Roberto; Cornette, Richard; Kikawada, Takahiro; Rebecchi, Lorena

Tardigrades are small micrometazoans able to resist several environmental stresses in any stage of their life cycle. An integrated analysis of tardigrade specimens collected in Tsukuba (Japan) revealed a peculiar morphology and a new sensory field in the cloaca. Molecular taxonomy and phylogenetic analysis on different genes (COI, ITS2, 18S and 28S) confirmed that this population is a new species, Macrobiotus kyoukenus sp. nov., belonging to the widespread Macrobiotus hufelandi group. The stress resistance capabilities of M. kyoukenus sp. nov. have been tested by submitting animals to extreme desiccation, rapid freezing, and high levels of ultraviolet radiations (UVB and UVC). Animals were able to survive desiccation (survivorship 95.71 ± 7.07%) and freezing up to -80 °C (82.33 ± 17.11%). Both hydrated and desiccated animals showed a high tolerance to increasing UV radiations: hydrated animals survived to doses up to 152.22 kJ m−2 (UVB) and up to 15.00 kJ m−2 (UVC), while desiccated specimens persisted to radiations up to 165.12 kJ m−2 (UVB) and up to 35.00 kJ m−2 (UVC). Present data contribute to the discovery of a larger tardigrade biodiversity in Japan, and the tolerance capabilities of M.

2021 - FCHgo: Fuel Cells Hydrogen educational model for schools, an imaginative approach to hydrogen and fuel cell technology for young students and their teachers [Articolo su rivista]
Dumont, E; Fuchs, H U; Corni, F; Contini, A; Altiero, T; Romagnoli, M; Karwasz, G P

In this paper we will describe didactic elements in the Horizon 2020 project FCHgo. This project is directed at children and adolescents between 8 and 18 years old. Its ultimate goal is to raise awareness for renewable energy sources, in particular hydrogen as a fuel and fuel cells for electric power. As part of the project, we are developing a toolkit for teachers and pupils, based upon a narrative approach to physical science and engineering. We believe the narrative approach to be best suited for this project because it allows us to take into account the cognitive tools available to pupils at various stages of their development.

2020 - Life-history traits and description of the new gonochoric amphimictic Mesobiotus joenssoni (Eutardigrada: Macrobiotidae) from the island of Elba, Italy [Articolo su rivista]
Guidetti, Roberto; Gneuß, Elisa; Cesari, Michele; Altiero, Tiziana; Schill, Ralph O

Comparative analyses of life-history theory studies are based on the characteristics of the life cycles of different species. For tardigrades, life-history traits are available only from laboratory cultures, most of which have involved parthenogenetic species. The discovery of a new gonochoristic bisexual Mesobiotus species in a moss collected on the island of Elba (Italy) provides us with the opportunity to describe Mesobiotus joenssoni sp. nov. and to collect data on the life-history traits of cultured specimens to increase our knowledge of the life-history strategies present in tardigrades. This new species is differentiated from all other species of the genus by the presence of granules (~1 µm in diameter) on the dorsal cuticle of the last two body segments, two large bulges (gibbosities) on the hindlegs and long, conical egg processes. The species exhibits sexual dimorphism in body length, with females being longer than males of the same age. The mean lifespan of specimens was 86 days, with a maximum of 150 days. The mean age at first oviposition was 19.8 days and the mean egg hatching time 15.4 days. The life-cycle traits correspond to those collected for the only other two macrobiotid species with gonochoric amphimictic reproduction examined so far.

2019 - Dormancy in Freshwater Tardigrades [Capitolo/Saggio]
Bertolani, Roberto; Guidetti, Roberto; Altiero, Tiziana; Nelson, Diane R.; Rebecchi, Lorena

For more than two centuries, tardigrades have been well known for their ability to undergo dormancy. However, this capability has been well studied mainly in the so-called limnoterrestrial species, i.e., in the species colonizing moist terrestrial habitats, such as mosses, lichens, and leaf litter. In these kinds of substrates, tardigrades are active only when a film of water is available around their body so in this condition they behave like aquatic animals. When the substrate dries or freezes, tardigrades achieve dormancy (quiescence) by entering cryptobiosis, specifically anhydrobiosis or cryobiosis, respectively. In freshwater habitats, both forms of cryptobiosis have been verified only in species able to live both in freshwater and terrestrial habitats. In the truly freshwater (or limnic) species, anhydrobiosis has not been verified, while cryobiosis has been confirmed in a few species. Another dormancy phenomenon bound to diapause is frequent in freshwater species: encystment (sometimes found even in limnoterrestrial species). The cyst state, which involves deep structural and physiological modifications, has been known from the beginning of the past century, but only recently has its morphology and inducing factors been studied in depth. Although data on molecular mechanisms allowing cryptobiosis are available, this information does not exist for encystment.

2019 - High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Guidetti, Roberto; Cesari, Michele; Bertolani, Roberto; Altiero, Tiziana; Rebecchi, Lorena

For many years, Paramacrobiotus richtersi was reported to consist of populations with different chromosome numbers and reproductive modes. To clarify the relationships among different populations, the type locality of the species (Clare Island, Ireland) and several Italian localities were sampled. Populations were investigated with an integrated approach, using morphological (LM, CLSM, SEM), morphometric, karyological, and molecular (18S rRNA, cox1 genes) data. Paramacrobiotus richtersi was redescribed and a neotype designed from the Irish bisexual population. Animals of all populations had very similar qualitative and quantitative characters, apart from the absence of males and the presence of triploidy in some of them, whereas some differences were recorded in the egg shell. All populations examined had the same 18S haplotype, while 21 haplotypes were found in the cox1 gene. In four cases, those qualitative characters were correlated with clear molecular (cox1) differences (genetic distance 14.6–21.8%). The integrative approach, which considered the morphological differences in the eggs, the reproductive biology and the wide genetic distances among putative species, led to the description of four new species (Paramacrobiotus arduus sp. n., Paramacrobiotus celsus sp. n., Paramacrobiotus depressus sp. n., Paramacrobiotus spatialis sp. n.) and two Unconfirmed Candidate Species (UCS) within the P. richtersi complex. Paramacrobiotus fairbanksi, the only ascertained parthenogenetic, triploid species, was redescribed and showed a wide distribution (Italy, Spain, Poland, Alaska), while the amphimictic species showed limited distributions. The difference in distribution between apomictic and amphimictic populations can be explained by the difference in the dispersal potentials associated with these two types of reproduction.

2018 - Reproduction, Development and Life Cycles [Capitolo/Saggio]
Altiero, Tiziana; Suzuki, Atsushi; Rebecchi, Lorena

In tardigrades reproduction occurs only through eggs, fertilized or unfertilized, and therefore only through gametes. Tardigrades exploit several reproductive modes, amphimixis, self-fertilization and thelytokous parthenogenesis (both apomixis and automixis). These modes are often in close relationship with the colonized environment. As regards sexuality, tardigrades can be gonochoristic (bisexual or unisexual) or hermaphroditic. The anatomy of the reproductive apparatus of males, females and hermaphrodites and the maturative patterns of male and female germinal elements are presented and discussed, as well as the ultrastructure of spermatozoa and eggs, including their phylogenetic implications. In addition, mating and fertilization patterns, embryonic and post-embryonic development, sexual dimorphism and parental care are considered and discussed. Finally, vegetative reproduction does not occur in tardigrades, and their capability to regenerate is limited to a physiological tissue restoration of a few cells.

2018 - Will the Antarctic tardigrade Acutuncus antarcticus be able to withstand environmental stresses related to global climate change? [Articolo su rivista]
Giovannini, Ilaria; Altiero, Tiziana; Guidetti, Roberto; Rebecchi, Lorena

Because conditions in continental Antarctica are highly selective and extremely hostile to life, its biota is depauperate, but well adapted to live in this region. Global climate change has the potential to impact continental Antarctic organisms because of increasing temperatures and ultraviolet radiation. This research evaluates how ongoing climate changes will affect Antarctic species, and whether Antarctic organisms will be able to adapt to the new environmental conditions. Tardigrades represent one of the main terrestrial components of Antarctic meiofauna; therefore, the pan-Antarctic tardigrade Acutuncus antarcticus was used as model to predict the fate of Antarctic meiofauna threatened by climate change. Acutuncus antarcticus individuals tolerate events of desiccation, increased emperature and UV radiation. Both hydrated and desiccated animals tolerate increases in UV radiation, even though the desiccated animals are more resistant. Nevertheless, the survivorship of hydrated and desiccated animals is negatively affected by the combination of temperature and UV radiation, with the hydrated animals being more tolerant than desiccated animals. Finally, UV radiation has a negative impact on the life history traits of successive generations of A. antarcticus, causing an increase in egg reabsorption and teratological events. In the long run, A. antarcticus could be at risk of population reductions or even extinction. Nevertheless, because the changes in global climate will proceed gradually and an overlapping of temperature and UV increase could be limited in time, A. antarcticus, as well as many other Antarctic organisms, could have the potential to overcome global warming stresses, and/or the time and capability to adapt to the new environmental conditions.

2015 - Distribution of Calcium and Chitin in the Tardigrade Feeding Apparatus in Relation to its Function and Morphology [Articolo su rivista]
Guidetti, Roberto; Bonifacio, Alois; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

The cuticular portion of the tardigrade feeding apparatus is a complex structure that can be schematically divided into four parts: a buccal ring, a buccal tube, a stylet system (formed by two piercing stylets, each within a stylet coat, and two stylet supports), and the lining of a myoepithelial sucking pharynx. To better understand the function and evolution of the feeding apparatus, the morpho-functional traits and chemical composition of the structures forming the feeding apparatuses of eight different species of tardigrades were analyzed. These eight species are representative of almost all main phylogenetic lineages of the phylum. The calcium and chitin in the feeding apparatus were examined by light microscopy (LM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), energy dispersive X-ray spectroscopy (EDX), and Raman microspectroscopy (Raman). In all species, the feeding apparatus had been subjected to biomineralization due to CaCO3 encrustations organized in the crystalline form of aragonite. Aragonite and chitin are present in different concentrations in the feeding apparatus according to the structures and species considered. Generally, where the structures are rigid there is more aragonite than chitin, and vice versa. The buccal tube and piercing stylets are rich in calcium, with the piercing stylets apparently composed exclusively of aragonite. In eutardigrades, chitin is in higher concentration in the structures subject to higher mechanical stresses, such as the crests of the buccal crown and the condyles of the stylet furca.

2015 - Il progetto "Piccoli Scienziati": un approccio narrativo alle scienze per insegnanti in servizio [Relazione in Atti di Convegno]
Corni, Federico; Giliberti, Enrico; Altiero, Tiziana

Questo articolo presenta i principali riferimenti teorici alla base del progetto, per poi approfondire gli aspetti metodologici e didattici, con particolare riferimento alla narrazione e alla struttura verticale del curricolo proposto; seguono esempi di attività tratte da un segmento del percorso e infine viene illustrata la modalità di formazione degli insegnanti, così come è stata messa a punto negli anni si sperimentazione del progetto.

2015 - Innovazione nella didattica delle scienze nella scuola primaria e dell’infanzia: al crocevia fra discipline scientifiche e umanistiche. Atti del Convegno (Modena-Reggio Emilia, 21-22 novembre 2014). Articoli selezionati [Curatela]
Corni, Federico; Altiero, Tiziana

Il convegno “Innovazione nella didattica delle scienze nella scuola primaria e dell’infanzia: al crocevia fra discipline scientifiche e umanistiche” è giunto alla sua quarta edizione, nel 2016, come occasione di proficuo confronto all’insegna della continuità tra le discipline di area umanistica e quelle di area scientifica e tra ricercatori ed insegnanti. Il tradizionale divario tra mondo accademico e mondo della scuola viene infatti in questo appuntamento messo in discussione: lo scambio di ricerche, pratiche didattiche e considerazioni trovano nel convegno sulla didattica delle scienze il luogo deputato al dialogo interdisciplinare e al confronto costruttivo. Il convegno anche quest’anno ha confermato il forte carattere multidisciplinare e ha portato all’attenzione del pubblico testimonianze e ricerche dal forte carattere interdisciplinare: le scienze naturali e le scienze umanistiche hanno cercato finalità, strumenti e metodologie condivisibili: mentre la terza edizione del convegno tenutasi nel 2014 ha voluto esplorare ed approfondire le storie e la narrazione come strumento di costruzione di significati e di stimolo allo sviluppo del pensiero formale a partire dal linguaggio naturale, il convegno del 2016 focalizza la sua attenzione sulla metafora e sul suo rapporto con la narrazione per l’educazione scientifica. Anche quest’anno il convegno è strutturato in tre sessioni. Nella prima sessione (2 dicembre 2016) e seconda (3 dicembre 2016, mattina), studiosi riconosciuti a livello internazionale, esperti in diversi settori della ricerca, da quella umanistica a quella scientifica, presentano aspetti teorici frutto di recenti studi riguardanti le scienze cognitive, linguistiche e pedagogiche che si rivelano sempre più fondamentali per la didattica delle scienze nella scuola primaria e dell’infanzia. Sono affrontati temi e problematiche riguardanti anche la didattica delle scienze e la formazione insegnanti, presentando sperimentazioni metodologiche di percorsi scientifici realizzati nelle scuole. La terza sessione (3 dicembre 2016, pomeriggio), come di consueto, è dedicata alle presentazioni e alla discussione di contributi riguardanti le esperienze di pratica didattica e di ricerca inerenti al tema del convegno da parte dei numerosi insegnanti in servizio e in formazione e dei ricercatori provenienti da diverse regioni italiane e da altri stati europei. Questo volume riporta contributi selezionati tra quelli presentati al convegno. La lingua è quella originale dell'autore per preservare l'autenticità e la fedeltà del contenuto.

2015 - Life history traits and reproductive mode of the tardigrade Acutuncus antarcticus under laboratory conditions: strategies to colonize the Antarctic environment [Articolo su rivista]
Altiero, Tiziana; Giovannini, Ilaria; Guidetti, Roberto; Rebecchi, Lorena

Global climate change has become an important issue, particularly for organisms living in the Antarctic region, as the predicted temperature increase can affect their life history traits. The reproductive mode and life history traits of one of the most widespread species of tardigrades in Antarctica were analyzed. Specimens of the eutardigrade Acutuncus antarcticus from a temporary freshwater pond at Victoria Land (Antarctica) were individually cultured. This species reproduced continuously by thelytokous meiotic parthenogenesis. Its life cycle was short (60–90 days) and the reproductive output was low, with a short generation time (25–26 days). The maternal effect can be responsible of the phenotypic plasticity observed in life history traits of the three analyzed generations that may be seen as a bethedging strategy, as also observed in other animals inhabiting stochastic environments. These traits, along with the cryptobiotic capability of A. antarcticus, are advantageous for exploiting the conditions suitable for growth and reproduction during the short Antarctic summer, and can explain its wide distribution on the Antarctic continent. These results open new avenues of research for determining the role of bet-hedging strategy in organisms living in unpredictable environments.

2015 - Space Flight Effects on Antioxidant Molecules in Dry Tardigrades: The TARDIKISS Experiment [Articolo su rivista]
Angela, Maria Rizzo; Altiero, Tiziana; Paola, Antonia Corsetto; Gigliola, Montorfano; Guidetti, Roberto; Rebecchi, Lorena

The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload, a set of multidisciplinary experiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in 2011. This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravity environment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space Station. TARDIKISS was composed of three sample sets: one flight sample and two ground control samples. These samples provided the biological material used to test as space stressors, including microgravity, affected animal survivability, life cycle, DNA integrity, and pathways of molecules working as antioxidants. In this paper we compared the molecular pathways of some antioxidant molecules, thiobarbituric acid reactive substances, and fatty acid composition between flight and control samples in two tardigrade species, namely, Paramacrobiotus richtersi and Ramazzottius oberhaeuseri. In both species, the activities of ROS scavenging enzymes, the total content of glutathione, and the fatty acids composition between flight and control samples showed few significant differences. TARDIKISS experiment, together with a previous space experiment (TARSE), further confirms that both desiccated and hydrated tardigrades represent useful animal tool for space research.

2014 - "Educare a pensare - Alberto Manzi", una mostra sul maestro Manzi e i suoi scritti naturalistici [Articolo su rivista]
Bisanti, Matteo; Pederzoli, Aurora; Altiero, Tiziana; Gambarelli, Andrea; Sabatini, Maria Agnese

Dal novembre 2012 al gennaio 2013, presso il Foro Boario di Modena, ha avuto luogo la mostra “Educare a pensare – Alberto Manzi” dedicata alla figura del maestro Alberto Manzi, divenuto famoso grazie alla trasmissione televisiva Rai degli anni ‘60 “Non è mai troppo tardi”. La mostra lo ha ricordato non solo per questo, ma ha preso in considerazione molti aspetti della sua personalità. Egli infatti fu anche biologo e amante della natura, tanto da dedicarsi nel 1955 a uno studio sulle formiche in Sudamerica, e fu autore di numerosi libri per ragazzi, come “Orzowei”, nonché di scrit- ti dedicati alla didattica. La mostra è nata dalla collaborazione tra il Centro Alberto Manzi di Bolo- gna, il Museo di Zoologia e Anatomia Comparata dell’Università degli Studi di Modena e Reggio Emilia e docenti e ricercatori dello stesso Ateneo. In particolare, il Centro Alberto Manzi ha curato la parte dedicata alla vita e alle opere di Alberto Manzi, l’Università di Modena e Reggio Emilia si è invece occupata dell’impegno del maestro nell’ambito dell’educazione e divulgazione scientifica, proponendo tra l’altro laboratori didattici ideati seguendo il pensiero di Manzi.

2014 - Biodiversity and adaptive strategy to Antarctica: the tardigrades [Abstract in Atti di Convegno]
Vecchi, Matteo; Mcinnes, Michele Cesari Sandra; Giovannini, Ilaria; Altiero, Tiziana; Rebecchi, Lorena; Bertolani, Roberto; Guidetti, Roberto

Tardigrades are important members of the Antarctic biota in terms of abundance, distribution, and colonized substrates. Despite their importance and regular occurrence in the harsh Antarctic terrestrial ecosystems, there have been few ecological or taxonomic studies. We carried out an extensive sampling campaign along Victoria Land coastal line, collecting 180+ samples of lichens, mosses and freshwater sediments over a c. 600 km North–South transect. These samples revealed an unexpectedly high diversity: 14 species, four of which new for science. Our results have underlined the level of undiscovered biodiversity in Antarctica. Some species had very localized distribution and occurred in specific substrates (e.g. moss or lichen). Other species were more scattered, and one (the endemic Acutuncus antarcticus) was present in almost all sampling areas and substrate types. This situation indicates that, as in temperate areas, Antarctica has tardigrade species with low dispersal capabilities, and a few species with high dispersal capabilities. Surprisingly, the genetic variability of A. antarcticus (COI gene) was extremely low even between very distant populations. Laboratory experiments on the adaptive strategy of A. antarcticus indicated a very short life cycle (c. 3-4 months), shorter than in species from temperate regions. Moreover, A. antarcticus exhibits thelytoky and has cryptobiotic capabilities. These attributes allowed this species to colonize almost all viable habitats in Antarctica

2014 - Innovazione nella didattica delle scienze nella scuola primaria e dell'infanzia: al crocevia fra discipline scientifiche e umanistiche. Atti del convegno 2012 - Articoli selezionati. [Curatela]
Corni, Federico; Altiero, Tiziana

La seconda edizione del convegno “Innovazione nella didattica delle scienze nella scuola primaria e dell’infanzia: al crocevia fra discipline scientifiche e umanistiche” che, nella sua prima edizione del 2010, nasceva in risposta all’esigenza di dialogo tra mondo della scuola e della ricerca didattica e tra discipline scientifiche e discipline umanistiche, si è realizzata nel 2012 per continuare a tenere vivo questo dialogo aprendosi anche alla scuola dell’infanzia. Il convegno è stato organizzato in tre sessioni. Nella prima sessione (30 novembre 2012), i relatori invitati hanno sviluppato e approfondito ulteriormente i temi aperti nella precedente edizione del 2010. Sono stati quindi presentati aspetti teorici frutto di recenti studi riguardanti le scienze cognitive, linguistiche e pedagogiche che si rivelano sempre più fondamentali per la didattica delle scienze nella scuola primaria e dell’infanzia. Nella seconda sessione (1 dicembre 2012, mattina), i relatori invitati hanno affrontato temi e problematiche riguardanti la formazione insegnanti, presentando sperimentazioni metodologiche di percorsi scientifici realizzati nelle scuole. Successivamente, una prima tavola rotonda intitolata “Le scienze per un curricolo verticale e interdisciplinare” ha consentito ai relatori invitati e a tutti i partecipanti di confrontarsi sulle tematiche presentate. La terza sessione (1 dicembre 2012, pomeriggio) è stata dedicata alle presentazioni delle esperienze di pratica didattica e dei dati di ricerca inerenti al tema del convegno dei numerosi insegnanti in servizio e in formazione e dei ricercatori provenienti da diverse regioni italiane e da altri stati europei. Le relazioni presentate hanno affrontato argomenti riconducibili a quattro tematiche principali: narrazione, infanzia, linguaggio e concetti elementari. Il convegno si è concluso con una tavola rotonda intitolata “Prospettive per un insegnamento interdisciplinare” presieduta dal Direttore del Dipartimento di Educazione e Scienze Umane dell’Università di Modena e Reggio Emilia, alla quale hanno partecipato anche ricercatori e docenti dello stesso Dipartimento che si sono confrontati sui temi del convegno.

2014 - Percorsi didattici museali: il caso di studio della sezione di educazione e divulgazione scientifica della mostra “Educare a pensare - Alberto Manzi” [Articolo su rivista]
Bisanti, Matteo; Razzoli, Joel; Gambarelli, Andrea; Altiero, Tiziana; Pederzoli, Aurora

Accanto alla mostra “Educare a pensare - Alberto Manzi”, dedicata alla vita e agli scritti di Alber- to Manzi, è stata avviata una ricerca per valutare l’efficacia comunicativa delle sale esposte. In par- ticolare ci si è focalizzati sulle sale dedicate agli ambienti naturali, allestite dal Museo di Zoologia e Anatomia Comparata dell’Università di Modena e Reggio Emilia e docenti e ricercatori dello stes- so Ateneo. Per fare questo si sono messe in campo varie metodologie di analisi: tracking dei visita- tori, osservazioni in incognito, questionari di gradimento somministrati al pubblico. Inoltre si è aggiunta anche un’analisi dei laboratori didattici proposti per la mostra grazie alla collaborazione di due classi della scuola primaria “L. Bassi” di Scandiano (RE) e due classi della scuola seconda- ria di primo grado “G. Marconi” di Modena. Lo scopo è stato quello di valutare l’apprendimento da parte degli alunni, ma anche di raccogliere informazioni per migliorare i laboratori didattici futuri.

2014 - Phylogeny of Eutardigrada: new molecular data and their morphological support lead to identification of new evolutionary lineages [Articolo su rivista]
Bertolani, Roberto; Guidetti, Roberto; Marchioro, Trevor; Altiero, Tiziana; Rebecchi, Lorena; Cesari, Michele

Seventy-nine specimens belonging to six of the nine current eutardigrade families have been considered in a wide and extensive study of the phylogeny of the largest class of Tardigrada (Eutardigrada). The molecular (18S rDNA) and morphological data partly support previous results. In particular, the applied integrative approach allowed us to find morphological synapomorphies, supporting the clusters here identified by molecular data and the previous taxa erected only on molecular basis. The class Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies Macrobiotoidea, Hypsibioidea and Isohypsibioidea, and all the families and subfamilies considered, even though in several cases with an emended diagnosis. In addition, new taxa have been erected: the superfamily Eohypsibioidea, the new families Hexapodibiidae and Isohypsibiidae, the new subfamily Pilatobiidae (Hypsibiidae) with the new genus Pilatobius, in addition to an upgrading to genus level of Diphascon and Adropion, to date considered subgenera of Diphascon. Our results demonstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative approach using molecular and morphological data is necessary to fully elucidate evolutionary relationships.

2014 - The short life cycle of Acutuncus antarcticus (Tardigrada) as adaptation to Antarctic environment [Abstract in Rivista]
Giovannini, Ilaria; Rebecchi, Lorena; Guidetti, Roberto; Altiero, Tiziana

The 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.

2014 - The toughest animals on the Earth: desiccation tolerance and oxidative stress in tardigrades. [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Giovannini, Ilaria; Guidetti, Roberto

Tardigrades 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.

2013 - Effetti delle radiazioni ultraviolette e della temperatura su un organismo della meiofauna antartica: Acutuncus antarcticus (Eutardigrada). [Abstract in Rivista]
Altiero, Tiziana; Guidetti, Roberto; Giovannini, Ilaria; Cesari, Michele; Montorfano, G.; Rizzo, A. M.; Bertolani, Roberto; Rebecchi, Lorena

I cambiamenti climatici in atto, oltre ad aumentare la temperatura, riducono la quantità di ozono troposferico. Questo porta ad un aumento delle radiazioni ultraviolette (UV), con potenziali conseguenze negative sulla salute umana e sugli ecosistemi. Tardigradi e rotiferi, principali componenti della meiofauna terrestre antartica, sono molto vulnerabili agli effetti sinergici di temperatura e UV, in quanto la loro stagione di sviluppo coincide con il periodo primaverile antartico, in cui normalmente si ha una diminuzione dell’ozono. Sono state quindi analizzate le risposte fisiologiche e biochimiche all’incremento di temperatura e UV nell’eutardigrado Acutuncus antarcticus, una delle specie più abbondanti nelle briofite e nei piccoli invasi di acqua dolce dell’Antartide. Esperimenti di essiccamento in condizioni controllate hanno evidenziato che A. antarcticus è in grado di attuare l’anidrobiosi, mostrando un’elevata sopravvivenza (92,8%), insolita per una specie di tardigrado generalmente dulciacquicola. Gli antiossidanti non sembrano però essere responsabili di tale sopravvivenza in quanto, ad eccezione della catalasi, non sono emerse differenze significative nell’attività/quantità di antiossidanti tra esemplari idratati e secchi, a differenza di quanto osservato in Paramacrobiotus richtersi, una specie di tardigrado prettamente “terrestre” che vive in zone temperate. Esperimenti tesi a valutare la resistenza di esemplari attivi di A. antarcticus all’incremento della temperatura (da 8°C a 41°C) hanno dimostrato che la specie è in grado di tollerare temperature elevate (a 33°C = 100% vivi; a 37°C = 35% vivi), anche se per breve tempo. Entrambi gli stati fisiologici (idratato ed essiccato) di A. antarcticus hanno dimostrato una buona resistenza alle radiazioni UV. Gli esemplari idratati hanno resistito fino alla dose di 61,9 kJ m-2 (5% vivi), mentre quelli in stato essiccato fino alla dose di 74,8 kJ m-2(7,5% vivi). Negli animali attivi, alla LD50 di 28,6 kJ m-2, l’effetto negativo degli UV aumenta in combinazione con l’incremento della temperatura (8°C: = 42,6% vivi; 15°C: = 1,7% vivi), dimostrando l’esistenza di un effetto sinergico. Pur restando in attesa di maggiori informazioni sul ciclo vitale, si può formulare l’ipotesi che questa specie antartica possieda ampie potenzialità di sopravvivenza ad eventuali cambiamenti ambientali.

2013 - Physiological and biochemical adaptive responses and defense mechanisms to withstand increasing ultraviolet radiation and temperatures in an Antarctic meiofauna organism. [Abstract in Atti di Convegno]
Giovannini, Ilaria; Guidetti, Roberto; Altiero, Tiziana; Cesari, Michele; Montorfano, G.; Rizzo, A. M.; Bertolani, Roberto; Rebecchi, Lorena

Physiological and biochemical adaptive responses and defense mechanisms to withstand increasing ultraviolet radiation and temperatures in an Antarctic meiofauna organism.

2013 - Tardigrades: multicellular organisms aboard the International Space Station (ISS) [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Guidetti, Roberto; Montorfano, G.; Cesari, Michele; Bertolani, Roberto; Rizzo, A. M.

Tardigrades: multicellular organisms aboard the International Space Station

2012 - BIOKIS: a model payload for multidisciplinary experiments in microgravity. [Articolo su rivista]
Vukich, M.; P. L., Ganga; D., Cavalieri; D., Rivero; S., Pollastri; S., Mugnai; S., Mancuso; S., Pastorelli; M., Lambreva; A., Antonacci; A., Margonelli; I., Bertalan; U., Johanningmeier; M. T., Giardi; G., Rea; M., Pugliese; M., Quarto; V., Roca; A., Zanin; O., Borla; Rebecchi, Lorena; Altiero, Tiziana; Guidetti, Roberto; Cesari, Michele; Marchioro, Trevor; Bertolani, Roberto; E., Pace; A., De Sio; M., Casarosa; L., Tozzetti; S., Branciamore; E., Gallori; M., Scarigella; M., Bruzzi; M., Bucciolini; C., Talamonti; A., Donati; V., Zolesi

In this paper we report about 1 the BIOKIS 2 payload: a multidisciplinary set of experiments and measurements in the fields of Biology and Dosimetry performed in microgravity. BIOKIS took advantage of the last STS-134 Endeavour mission and engineering state of the art in Space Life Science. The BIOKIS payload is compact, efficient, and capable to host experiments with different samples and science disciplines. Moreover, the time overlap of biological experiments and dosimetry measurements will produce more insightful information.

2012 - Comparative analyses of the cuticular and muscular structures of the buccal-pharyngeal apparatus of tardigrades [Abstract in Atti di Convegno]
Guidetti, Roberto; Marchioro, Trevor; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

In spite of the great importance of the tardigrade buccal pharyngeal apparatus in taxonomic and phylogenetic studies, it received little attention as regards its evolution and operating mechanism. To understand the relationships between form and function of the structures acting in the functioning of buccal-pharyngeal apparatus (i.e. cuticular structures, muscular fibers, pharynx), and to increase our knowledge on this apparatus, a comparative analyses using different investigation techniques was performed. The buccal-pharyngeal apparatuses of three species have been studied, Echiniscus trisetosus, Milnesium tardigradum e Paramacrobiotus richtersi, as representative of the two classes and of three orders of tardigrades. The cuticular structures of the buccal-pharyngeal apparatus have been analyzed form a morphological (light microscopy, scanning electron microscopy – SEM, and confocal laser scanner microscopy -CLSM) and chemical (energy dispersive X-ray spectroscopy) point of views. The musculature associated to the sclerified structures of the buccal-pharyngeal apparatus has been analyzed by CLSM to identify the muscular fibers and their relationships with the sclerified structures. The differences in the general anatomy of the buccal-pharyngeal apparatus among the three species were high, even though homologous structures were recognizable. The higher differences among species were found in the organization of the muscular system responsible of the stylet movements. The detailed analyses of the buccal-pharyngeal apparatus allowed a new interpretation of the organization of the stylet systems, and to understand the muscular system related to the feeding. The chemical analyses showed that the piercing stylets were formed by calcium, in form of CaCO3. Heterotardigrada were differentiated from Eutardigrada for the presence of high concentration CaCO3 encrustations in the buccal tube. Within Eutardigrada, Apochela differs from Parachela since they are characterized by the absence of CaCO3 in the buccal tube.

2012 - Diversified bet-hedging strategy in the desiccation tolerant tardigrade Paramacrobiotus richtersi [Abstract in Atti di Convegno]
Altiero, Tiziana; Rossi, V.; Bertolani, Roberto; Rebecchi, Lorena

In variable environmental conditions a genotype may reduce the risk associated with reproduction by two main types of bet-hedging strategies: conservative and diversified bet-hedging. The former involves an avoidance of extremes to minimize fluctuations in a life-history trait that will assure an optimal mean for energetic constraints. The latter entails probabilistic risk spreading among individuals of the same genotype that express a range of diversified phenotypes to sample a range of different environments through time. Diversified bet-hedging is generally considered the most viable strategy for a long-term performance in a habitat varying in an unpredictable way. To date, only a few empirical studies provide evidence that bet-hedging occurs in nature. The timing of many phenological events (e.g. egg hatching) results from a complex interplay among organism genotype, environmental factors and maternal effects (e.g. egg size). The relationship between egg size and development time is known and it is generally accepted that larger eggs take longer to develop than smaller ones. The production of eggs with variable size may represent a strategy by which a mother spreads the risk connected with life in a temporary habitat. As regards tardigrades, they evolved a large variety of dormant stages that can be ascribed to diapause (encystment, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). Therefore, their life-cycle can be generally divided into two very distinct stages: the active stage and the dormant stage, which are characterized by substantially different requirements and risks. During the active stage, tardigrades may exhibit high plasticity in life history traits (e.g. egg number, egg size and hatching time), as an adaptive measure to cope with risks linked to unpredictable habitat conditions. Clonal lineages from an apomictic population of Paramacrobiotus richtersi (Eutardigrada, Macrobiotidae) may produce up to four kinds of eggs: subitaneous eggs, delayed-hatching eggs, abortive eggs and diapause resting eggs, the last ones require a stimulus to hatch (e.g. rehydration after a period of desiccation). The lack of genetic variation expected among clonal organisms make them ideal material for investigating diversified bet-hedging that is, by definition, life history trait variance expressed within genotypes, and maternal effects. We compared the proportion of different kinds of eggs and analysed the correlation between egg size and hatching time within clonal eggs. Our preliminary results provide possible empirical support of the occurrence of diversified bet-hedging strategy due to maternal effect in tardigrades living in stochastic environments.

2012 - Form and function of the feeding apparatus in Eutardigrada (Tardigrada) [Articolo su rivista]
Guidetti, Roberto; Altiero, Tiziana; Marchioro, Trevor; L., Sarzi Amadè; A. M., Avdonina; Bertolani, Roberto; Rebecchi, Lorena

Tardigrade feeding apparatus is a complexstructure with considerable taxonomic significance that canbe schematically divided into four parts: buccal ring, buccaltube, stylet system, and pharynx. We analyzed the finemorphology and the tridimensional organization of thetardigrade buccal–pharyngeal apparatus in order to clarifythe relationships between form and function and to identifynew characters for systematic and phylogenetic studies. Weconducted a comparative analysis of the cuticular structuresof the buccal–pharyngeal apparatuses of twelve eutardigradespecies, integrating data obtained by SEM and LMobservations. Morphological diversity was observed andnew cuticular structures such as the stylet coat of the styletsystem were identified. The synthesis of the buccal–pharyngealapparatus during molting was also analyzedobtaining a clear developmental sequence of its resynthesis.These findings lead us to redefine the previous interpretationsof the functioning mechanisms of the buccal–pharyngealapparatus and provide a more specific relationship between tardigrade diet and the anatomy of their feedingapparatuses. In addition, the detection by energy-dispersiveX-ray spectroscopy of calcium in the stylets, buccal tube,and placoids of eutardigrade species (i.e., Milnesium tardigradum,Paramacrobiotus richtersi) indicates that CaCO3incrustations are not an exclusive feature of heterotardigradesand lead to suppose that this trait was present in theancestors of both classes.

2012 - Gli animali de "Le avventure di Pinocchio" [Articolo su rivista]
Altiero, Tiziana; Rebecchi, Lorena

The aim of this paper is to increase our knowledge on the about 60 different kind of animals cited in the literary work “Le avventure di Pinocchio”. Through brief and fleeting apparitions, these animals have the task of supporting the symbolic language of the narrative because advisors, guides, barriers and helpers of Pinocchio, or as caricature of the institutions. The knowledge of the animals diversity characterizing the “Le avventure di Pinocchio” is analyzed considering their geographical distribution and colonized habitats. Moreover, the metaphoric meaning of the animals is also considered.

2012 - Heat-shock protein in encysted and anhydrobiotic eutardigrades [Articolo su rivista]
Altiero, Tiziana; Guidetti, Roberto; D., Boschini; Rebecchi, Lorena

The Heat 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.

2012 - Identification of the trehalose-6-phosphate synthase (tps) gene in desiccation tolerant and intolerant tardigrades [Articolo su rivista]
Cesari, Michele; Altiero, Tiziana; Rebecchi, Lorena

The presence of the gene coding for trehalose-6-phosphate synthase (tps) was determined for the first time in Tardigrada in twelve species belonging to both Heterotardigrada and Eutardigrada. The tps gene was found in all semiterrestrial and anhydrobiotic species examined, correlating trehalose presence with anhydrobiotic ability. In contrast, tps was not detected in two limnic and desiccation intolerant species. The surprising presence of tps in another limnic and desiccation intolerant species, Dactylobiotus parthenogeneticus, allowed us to infer that in tardigrades trehalose could be produced and involved, not just in anhydrobiosis, but also in the regulation of other biological functions, such as encystment. Our findings, integrated with literature data, indicate that anhydrobiotic ability in tardigrades can depend on the combined action of numerous different molecules, only partially identified. With nucleotide tps sequences, it is impossible to infer phylogenetic relationships inside the phylum Tardigrada, while amino acid sequences further support the relationship between Tardigrada and Pancrustacea.

2012 - L'approccio di DNA barcoding nello studio dei tardigradi [Abstract in Atti di Convegno]
Bertolani, Roberto; Rebecchi, Lorena; Guidetti, Roberto; Altiero, Tiziana; Giovannini, Ilaria; Mori, L.; Cesari, Michele

Il DNA barcoding è stato proposto da Hebert e coll. nel 2003 con lo scopo di discriminare entità biologiche attraverso l’analisi di un singolo locus genico. Oggi il suo utilizzo appare in rapida ascesa (più di 450 lavori pubblicati riguardanti organismi anche molto differenti). Ciononostante, esistono ancora molte controversie sull’utilizzo di questo approccio in tassonomia. È importante sottolineare che solitamente il DNA barcoding non dovrebbe essere impiegato per la filogenesi, anche se a volte ciò è accaduto, ma piuttosto focalizzarsi sulla costruzione di una chiave di identificazione molecolare universale, basata però su consistenti informazioni tassonomiche che devono essere incluse nella barcode reference library. Nel Phylum Tardigrada, il DNA barcoding rappresenta un approccio recente per l’identificazione delle specie e per la risoluzione di problemi tassonomici, anche alla luce delle ridotte dimensioni degli animali e della limitatezza dei caratteri morfologici disponibili. Da queste premesse è stato sviluppato il progetto MoDNA (Morfologia e DNA), combinando lo studio di un frammento del gene mitocondriale citocromo c ossidasi I (cox1) con dati morfologici anche a livello fine, allo scopo di sviluppare e validare l’integrative taxonomy sul modello tardigradi. L’indagine è stata condotta su gruppi di specie affini e criptiche in più famiglie di Eutardigrada. La costruzione di un database di sequenze di riferimento è di importanza capitale per una corretta applicazione di questa metodica. Tuttavia, non è meno importante che queste sequenze siano strettamente correlate a specifici voucher specimens validati da esperti tassonomi. In mancanza di questo, il database di riferimento non può essere del tutto affidabile. Per raggiungere questo importante obiettivo sono stati sviluppati metodi e protocolli per ottenere risultati standardizzati ed una stretta corrispondenza tra sequenza di DNA e morfologia, possibilmente con documentazione sia al microscopio ottico che elettronico a scansione. Un valore aggiunto è rappresentato dalla possibilità di utilizzo di materiale proveniente dal locus typicus di una specie già descritta e ancor più dall’utilizzo del DNA barcoding nella descrizione di nuove specie.

2012 - Nature, Source and Function of Pigments in Tardigrades: In Vivo Raman Imaging of Carotenoids in Echiniscus blumi [Articolo su rivista]
A., Bonifacio; Guidetti, Roberto; Altiero, Tiziana; V., Sergo; Rebecchi, Lorena

Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.

2012 - Raman imaging study on living tardigrades: origin, nature and function of pigments in Echiniscus blumi [Abstract in Atti di Convegno]
Bonifacio, A.; Guidetti, Roberto; Altiero, Tiziana; V., Sergo; Rebecchi, Lorena

Tardigrades exhibit a variety of pigmentations. Several species are translucent or whitish, but many others are markedly colored with red-orange, brown, green and yellow pigments. These pigments may be found in the body cavity, storage cells, epidermis, eye-spots and, according to literature, in cuticle. The genus Echiniscus is composed of many species. Despite occurrence of pigments is well documented for many of them, the chemical nature, source and function (especially in relation to their resistance to harsh physical and chemical conditions) of these pigments remain unknown. Some hypotheses were formulated about them, but to the best of our knowledge no direct and conclusive experimental proof has been reported to date. Therefore, we are taking this opportunity to attempt a resolution for these open questions using Raman spectroscopy on living individuals of Echiniscus blumi. Raman spectroscopy is a non-destructive and (semi)-quantitative analytical technique based on scattering laser radiation by vibrating molecules, which proved to be an ideal tool for studying living cells and biological tissues. Moreover, by incorporating Raman micro-spectroscopy it is possible to obtain an image of the spatial distribution of the main biochemical constituents of a biological sample (i.e. Raman mapping or imaging). In our analyses, pigments in E. blumi are identified as carotenoids. Their spectra well match those available in literature for β-carotene, zeaxanthin and β-cryptoxanthin, all having 11 conjugated C=C bonds, but not that of lutein, which has only 10. Previously reported chemical reactivity data of Echiniscus pigments suggest β-carotene as the most likely candidate pigment in the genus. Moreover, pigment distribution within the animal body cavity is imaged with Raman mapping. The dietary origin of the pigments (from the moss, Grimmia orbicularis) is demonstrated, as well as their presence in the eggs and in eye-spots, and their absence in the animal cuticle. Using in-vivo Raman imaging, a decrease in carotenoid content is detected after the induction of oxidative stress on animals, supporting the hypothesis of an antioxidant function of these pigments during anhydrobiosis. Considering the lack of methods to directly study antioxidant function of carotenoids in vivo, pigmented tardigrades, investigated with Raman imaging, could be used as model organisms for this purpose opening new perspectives of research in living organisms.

2012 - Systematic position of Macrobiotus glebkai within the “hufelandi group”, based on morphology and molecular analysis of a population from Ukraine [Abstract in Atti di Convegno]
Kiosya, Y. e. O.; Bertolani, Roberto; Rebecchi, Lorena; Altiero, Tiziana; Cesari, Michele

The “Macrobiotus hufelandi group” is characterized by species which share some distinguishing animal and egg shell characters. In general, they have two rod-shaped macroplacoids and an evident microplacoid, eye spots, pores on the cuticle, medium sized claws, egg shell with inverted goblet-like processes, and spermatozoa with corkscrew-shaped head. We had the opportunity to analyse one species, Macrobiotus glebkai Biserov, 1990, found in Eastern Ukraine, which shows characters of the group in the animals, but a peculiar egg shell morphology. We have carried out a phylogenetic analysis on the 18S nuclear gene in order to verify the systematic position of this species. This analysis has been followed by an integrated study on morphology by LM and SEM, sex ratio, karyology and DNA barcoding (mtDNA cox1). The phylogenetic tree based on 18S sequences grouped M. glebkai with M. hufelandi and other related species with inverted goblet-like processes on the egg shell. This evidences that the egg shell morphology can sometimes be more variable than that of the animals. In depth morphological analysis allowed us to define details of both animals and eggs of M. glebkai, which showed the same characters as the Russian type material (that we also investigated). We verified that the population from Eastern Ukraine was composed by females and males and that the spermatozoon morphology is in line with that found in the various species of the “Macrobiotus hufelandi group”. The oocytes contain bivalents, but their number has to be specified, as oocytes of some specimens appeared to contain more than 6 bivalents (6 bivalents is the usual pattern for amphimictic species of the “M.hufelandi group”). The DNA barcoding has evidenced the presence of three haplotypes (Kimura 2-parameter distances: 0.2-0.3%) belonging to the same haplogroup, well differentiated (more than 20%) from M. hufelandi and the other considered species of the group.

2012 - Tardigrades on board of the STS-134 space flight [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Rizzo, A. M.; Cesari, Michele; Montorfano, G.; Marchioro, Trevor; Bertolani, Roberto; Guidetti, Roberto

The joint ASI-AM DAMA mission supported a set of scientific experiments executed in short-duration microgravity on board the Space Shuttle docked to the International Space Station. BIOKIS payload (by Kayser Italia) took advantage of the last STS-134 Endeavour mission lasted 16 days. It hosted a multidisciplinary set of experiments in the field of biology and dosimetry. Among them, the TARDIKISS project investigated the responses of live desiccated tardigrades, multicellular heterotrophic organisms, under space stresses. In particular, specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. The experimental flight units were hosted into the Biokon, a standard transportable container. In addition, a Laboratory Control sample was maintained in Modena laboratory for the duration of the flight, while a post-flight Temperature Control sample was exposed to the temperature profile experienced by tardigrades during the mission. For both species, the Flight animals did not show differences in survival with regard to Laboratory and Temperature control animals. Only in R. oberhaeuseri differences have been recorded between Flight and Temperature Control samples. Specimens of P. richtersi from Flight, Laboratory and Temperature control samples have been reared in lab. Flight females laid eggs with normal shape; several eggs have been able to hatch, and newborns exhibited, when adult, normal capability to reproduce. The comparative analysis of the antioxidant metabolism between Flight and Temperature control samples did not evidenced significantly differences between the two groups. These data confirm that tardigrades represent a useful animal tool for space research. The authors thanks the Italian Space agency, Italian Air Force and Kayser Italia S.r.l.

2012 - Two tardigrade species on board of the STS-134 space flight [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Rizzo, A. M.; Cesari, Michele; Montorfano, G.; Marchioro, Trevor; Bertolani, Roberto; Guidetti, Roberto

The TARDIKISS project (Tardigrades in Space) investigated the physiological and molecular effects of space stresses on alive desiccated tardigrades, as representative of multicellular organisms. It has been a part of the BIOKIS (Biokon in Space), a set of multidisciplinary experiments of DAMA (DArk MAtter) mission on board of STS-134 space flight, the last of the shuttle Endeavour, in the frame of a joint between ASI and Italian Air Force. In TARDIKISS, experimentally desiccated (anhydrobiotic) specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. Both species have very good anhydrobiotic ability, but differ for several biological and ecological characters. The experiment units of TARDIKISS were hosted into the Biokon, a standard transportable container designed and manufactured by Kayser Italia. In May 2011, the Biokon containing the TARDIKISS experiment unit was integrated on Middeck Locker of ISS and had flown for 16 days at Low Earth Orbit. Two sample sets were used as controls: the former (Laboratory Control) was maintained in Modena laboratory for the duration of the flight, and the latter one (Temperature Control) was a post-flight control in which samples were exposed to the temperature profile experienced by tardigrades the days immediately before, during, and just after the flight mission. For both species, the flight animals did not show differences in survival with regards of Laboratory control and Temperature control animals. Only in R. oberhaeuseri differences have been recorded between Flight and Temperature Control samples. Live specimens of P. richtersi from Flight samples, Laboratory control and Temperature control samples have been reared in lab. Flight females laid eggs with normal shape; several eggs have been able to hatch, and newborns exhibited normal morphology, behaviour and capability to reproduce. Moreover, a comparative analyses of the antioxidant metabolism between Flight samples and Temperature control samples has been done. No differences have been evidenced between the two groups, with the exception of the reductase activity: significant differences in reductase activity between Flight and Temperature control were evidenced (p<0.05) in R. oberhaeuseri. These first results lead us to deduce that during the DAMA mission, microgravity and cosmic radiations did not significantly affect survival of flown tardigrades, confirming that tardigrades represent a useful animal tool for space research.

2012 - What can we learn from the toughest animals of the Earth? Water bears (tardigrades) as multicellular model organisms in order to perform scientific preparations for lunar exploration. [Articolo su rivista]
Guidetti, Roberto; A. M., Rizzo; Altiero, Tiziana; Rebecchi, Lorena

Space missions of long duration required a series of preliminary experiments on living organisms, validated by a substantial phase of ground simulation experiments, in the field of micro- and intermediate gravities, radiobiology, and, for planetary explorations, related to risks deriving from regolith and dust exposure. In this review, we present the tardigrades, whose characteristics that recommend them as an emerging model for space biology. They are microscopic animals but are characterized by a complex structural organization similar to that of larger animals; they can be cultured in lab in small facilities, having small size; they are able to produce clonal lineages by means of parthenogenesis; they can completely suspend their metabolism when entering in dormant states (anhydrobiosis induced by dehydration and cryobiosis induced by freezing); desiccated anhydrobiotic tardigrades are able to withstand chemical and physical extremes, but a large tolerance is showed also by active animals; they can be stored in dry state for many years without loss of viability. Tardigrades have already been exposed to space stressors on Low Earth Orbit several times. The relevance of ground-based and space studies on tardigrades rests on the presumption that results could suggest strategies to protect organisms, also humans, when exposed to the space and lunar environments.

2011 - Con … tatto a pelle e orecchie tese. Progetto didattico di Biologia per la Scuola Primaria [Abstract in Atti di Convegno]
Altiero, Tiziana; Telleri, F.

La strategia di apprendimento per scoperta consente al bambino di imparare facendo (ricerca-azione). Partendo dal percorso “Con … tatto a pelle” del Museo di Zoologia e Anatomia Comparata (Università di Modena e Reggio Emilia), è stato progettato un percorso didattico per stimolare i bambini all’utilizzo degli organi di senso per scoprire gli animali. Scatole tattili, contenenti materiali di origine animale (pellicce, penne, zoccoli, lana grezza, pennelli, cinture, ecc.) e postazioni sonore hanno guidato i bambini di tre classi terze di una scuola primaria di Modena in un percorso sensoriale di scoperta di alcune caratteristiche dell’apparato tegumentario e delle strategie di comunicazione degli animali. E’ stato evidenziato un buon livello di partecipazione e di coinvolgimento emotivo degli alunni alle attività del progetto. Questo clima positivo ha favorito l’acquisizione di conoscenze sull’argomento oggetto di studio e la possibilità concreta di giungere a concetti generali (Mammiferi e Uccelli), a partire da quelli particolari (annessi cutanei). Il progetto didattico “Con … tatto a pelle e orecchie tese” rappresenta un buon esempio di applicazione del sistema formativo integrato, in cui la scuola e l’extra-scuola (Museo) possono essere un connubio costruttivo per un apprendimento significativo della biologia fin dalla scuola primaria.

2011 - DNA barcoding e strategie riproduttive nel genere Ramazzottius (Tardigrada, Eutardigrada) [Abstract in Atti di Convegno]
Rebecchi, Lorena; Bigi, Mila; Bertolani, Roberto; Altiero, Tiziana; Cesari, Michele

Nell’ambito del progetto MoDNA (morfologia e DNA) è stato utilizzato l’approccio del DNA barcoding, associato a comparazioni morfologiche e cariologiche. E’ stata valutata la variabilità inter- ed intraspecifica nel genere Ramazzottius, caratterizzato dalla presenza di specie criptiche e da diverse modalità riproduttive e talvolta diverso grado di ploidia. Mediante approccio morfologico (LM, SEM) e molecolare (mtDNA cox1) sono stati analizzati esemplari di Ramazzottius provenienti da 15 località europee. Per correlare morfologia e sequenze sono stati allestiti voucher specimens, compresi gusci di uova (molto importanti per la diagnosi specifica) dalle quali sono sgusciati neonati utilizzati per l’analisi molecolare. Tre campioni contengono rispettivamente Ramazzottius varieornatus, Ramazzottius tribulosus ed una nuova specie di Ramazzottius, distinguibili sia su base morfologica che molecolare. Negli altri campioni gli esemplari (animali e uova) sono tutti morfologicamente attribuibili a Ramazzottius oberhaeuseri, ma si distinguono tra loro per sex ratio, ploidia e per notevoli differenze molecolari. I dati evidenziano la validità dell’approccio integrato nello studio della diversità nei tardigradi e dell’utilizzo del DNA barcoding. Nel genere Ramazzottius, ed in particolare nell’ambito della morfospecie R. oberhaeuseri, è confermata la presenza di specie criptiche, alcune delle quali con ampia diffusione e con strategie riproduttive diverse.

2011 - On dormancy strategies in tardigrades. [Articolo su rivista]
Guidetti, Roberto; Altiero, Tiziana; Rebecchi, Lorena

In this review we analyze the dormancy strategies of metazoans inhabiting “hostile to life” habitats, which have a strong impact on their ecology and in particular on the traits of their life history. Tardigrades are here considered a model animal, being aquatic organisms colonizing terrestrial habitats. Tardigrades evolved a large variety of dormant stages that can be ascribed to diapause (encystment, cyclomorphosis, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). In tardigrades, diapause and cryptobiosis can occur separately or simultaneously, consequently the adoption of one adaptive strategy is not necessarily an alternative to the adoption of the other. Encystment and cyclomorphosis are characterized by seasonal cyclic changes in morphology and physiology of the animals. They share several common features and their evolution is strictly linked to the molting process. A bet-hedging strategy with different patterns of egg hatching time has been observed in a tardigrade species. Four categories of eggs have been identified: subitaneous, delayed-hatching, abortive and diapause resting eggs, which needs a stimulus to hatch (rehydration after a period of desiccation). Cryptobiotic tardigrades are able to withstand desiccation (anhydrobiosis) and freezing (cryobiosis) at any stage of their life-cycle. This ability involves a complex array of factors working at molecular (bioprotectans), physiological and structural levels. Animal survival and the accumulation of molecular damage are related to the time spent in the cryptobiotic state, to the abiotic parameters during the cryptobiotic state, and to the conditions during initial and final phases of the process. Cryptobiosis evolved independently at least two times in tardigrades, in eutardigrades and in echiniscoids.Within each evolutionary line, the absence of cryptobiotic abilities is more related to selective pressures to local habitat adaptation than to phylogenetic relationships. The selective advantages of cryptobiosis (e.g. persistency in “hostile to life” habitats, reduction of competitors, parasites and predators, escaping in time from stressful conditions) could explain the high tardigrade species diversity and number of specimens found in habitats that dry out compared to freshwater habitats.

2011 - Resistance of the anhydrobiotic eutardigrade Paramacrobiotus richtersi to space flight (LIFE–TARSE mission on FOTON-M3). , [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; Cesari, Michele; Bertolani, Roberto; A. M., Rizzo; P., Corsetto; Guidetti, Roberto

The project “TArdigrade Resistance to Space Effects (TARSE)” analyzed the effects of space environment stresses on the eutardigrade Paramacrobiotus richtersi allowing, for the first time, a comparison between hydrated and desiccated animals. TARSE experiments were part of the mission LIFE on FOTON-M3 that flew at an altitude between 250-290 km for 12 days in September 2007. In this paper, data on survival rate, Hsps expression (70 kDa and 90 kDa) and genomic DNA integrity (double strand breaks) of flown desiccated animals, and from specimens in ground control experiments are presented. For the flown experiments, microcosms of dry leaf litter collected in the field containing naturally desiccated specimens and square papers containing specimens desiccated under controlled conditions were used. After the space flight, desiccated animals had high survival rates (from 79% to 95%) showing a similar survival rate to that observed in ground controls. No visible damages to double strand genomic DNA were observed in all desiccated samples, while spaceflight induced the Hsps expression (Hsp70 and Hsp90) compared to ground controls. These data combined with earlier published results on hydrated specimens of P. richtersi indicate that tardigrades have the ability to tolerate for a short period of time several extreme stresses typical of the extraterrestrial environment.

2011 - Strutture cuticolari e muscolatura dell’apparato bucco-faringeo dei tardigradi [Abstract in Atti di Convegno]
Guidetti, Roberto; Marchioro, Trevor; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

L’apparato bucco-faringeo dei tardigradi è composto da parti cuticolari sclerificate (compresi due stiletti perforanti ritenuti una delle apomorfie del phylum) e muscolatura. Con questo studio si è voluto sia comprendere al meglio il meccanismo di funzionamento di questo apparato che individuare nuovi caratteri per gli studi di tassonomia e filogenesi. L’apparato bucco-faringeo di Echniscus trisetosus, Milnesium tardigradum e Paramacrobiotus richtersi (appartenenti a due classi e tre ordini del phylum) è stato studiato morfologicamente (microscopia ottica, microscopia elettronica a scansione) e chimicamente (spettroscopia a raggi X). E’ stata inoltre analizzata l’anatomia della muscolatura associata all’apparato mediante microscopia confocale a scansione laser. Nelle tre specie le differenze nell’anatomia dell’apparato bucco-faringeo risultano notevoli , così come quelle relative all’organizzazione dei fasci muscolari ad esso associati. L’analisi chimica ha rivelato che gli stiletti sono costituiti da CaCO3 e che le due classi di tardigradi si differenziano per la presenza/assenza di CaCO3 sul tubo boccale. L’analisi dettagliata delle strutture cuticolari dell’apparato bucco-faringeo e della sua muscolatura ha consentito una nuova interpretazione dell’organizzazione e del funzionamento del sistema di protrazione e retrazione degli stiletti. Sebbene nelle strutture sclerificate dei tre ordini siano riconoscibili diverse omologie, queste sono più difficilmente riscontrabili nella muscolatura.

2011 - Survival of freezing by hydrated tardigrades inhabiting terrestrial and freshwater habitats [Articolo su rivista]
Guidetti, Roberto; Altiero, Tiziana; Bertolani, Roberto; Grazioso, Pasqualina; Rebecchi, Lorena

The seasonality and unpredictability of environmental conditions at high altitudes and latitudes govern the life cycle patterns of organisms giving rise to stresses that cause dead or development of specific adaptations. Ice formation is a major variable affecting survival of both freshwater fauna and fauna inhabiting lichens, mosses and leaf litter. Tardigrades occupy a wide range of niches in marine, freshwater and terrestrial environments. The highest number of species is found in terrestrial habitats thanks to their ability to enter anhydrobiosis and cryobiosis. The cryobiotic ability of tardigrade species from polar regions is well known. Consequently, we focused our research on the ability to survive freezing in active hydrated state using seven tardigrade species differing in phylogenetic position and collected at various altitudes and from different habitats in a temperate area. Replicates were cooled at different cooling rates (from 0.31 °C min-1 to 3.26 °C min-1). Even though the final survival and the time required by animals to recover active life were both inversely related to the cooling rates, highly significant interspecific differences were found. Species survival ability ranges from excellent to none. Species living in xeric habitats withstand freezing better than those living in hygrophilous habitats, while true limnic species do not exhibit cryobiotic capabilities. The ability to withstand freezing seems linked to the anhydrobiotic ability. The difference in cryptobiotic performance among tardigrade species seems more influenced by selective pressures linked to local adaptation to habitat characteristics than by phylogenetic relationships.

2011 - TARDIKISS: tardigrades in the mission STS-134, the last of the shuttle Endeavour. [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Cesari, Michele; Marchioro, Trevor; Rizzo, A. M.; Ganga, P. L.; Vukich, M.; Donati, A.; Zolesi, V.; Bertolani, Roberto; Guidetti, Roberto

The TARDIKISS project (Tardigrades in Space) investigates the physiological and molecular effects of space stresses on alive desiccated multicellular organisms, the tardigrades. It is a part of the BIOKIS (Biokon in Space), a set of multidisciplinary experiments of DAMA (DArk MAtter) mission on board of STS-134 space flight, the last of the shuttle Endeavour, in the frame of a joint between ASI and Italian Air Force. In TARDIKISS, experimentally desiccated (anhydrobiotic) specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. Both species have very good anhydrobiotic ability, but differ for several biological and ecological characters. Paramacrobiotus richtersi is carnivorous, soil-dwelling and white in colour, while R. oberhaeuseri is lichen-dwelling, herbivorous, and characterized by a brown/red pigmentation. The experiment units of TARDIKISS were hosted into the Biokon, a standard transportable container designed and manufactured by Kayser Italia. In May 2011, the Biokon containing the TARDIKISS experiment unit was integrated on Middeck Locker of ISS and had flown for 16 days. As control, ground experiments replicating the temperature profile recorded within the Biokon during the flight are currently running. To this day, we have completed the analysis on tardigrade survival after the space flight and obtained preliminary data on life cycle parameters. Both tardigrade species showed a very high survival (98.33% for P. richtersi and 90.0% for R. oberhaeuseri) and no significant differences were scored between flight or Earth tardigrades. After the flight, specimens of P. richtersi were reared in lab. Females laid eggs with normal shape; several eggs hatched with a short hatching time and newborns exhibited normal morphology and behaviour. In the next step, genomic DNA quality and oxidative metabolism of flown tardigrades vs ground control tardigrades will be analysed.

2011 - The ultrastructure of the tardigrade spermatozoon: a comparison between Paramacrobiotus and Macrobiotus species (Eutardigrada, Macrobiotidae). [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; A., Guidi

The spermatozoan ultrastructure was investigated in three semiterrestrial eutardigrade species belonging to two different genera of Macrobiotidae (Paramacrobiotus and Macrobiotus). The spermatozoa of P. areolatus and P. richtersi are very similar and are made by three regions, namely a long head, a short kidney-shaped middle piece and a short tail with its terminal tuft. In both species the spermatozoa are particularly long (up to 100 µm) and very thin. The peculiar length is due to the remarkably developed head consisting of a cylindrical acrosome and a weakly coiled nucleus increasing in width caudally. The presence of a long nucleus, an electron-dense core of fibrils running parallel to the nucleus, as well as nine outer electron-dense fibers around the proximal part of the axoneme represents novelties in the ultrastructure of the tardigrade spermatozoa. These structures, never described before for a tardigrade spermatozoon, could be related to the movement of those extraordinary long male gametes of Paramacrobiotus. The spermatozoon of M. harmsworthi too is made up of three regions: the head, including the acrosome and nuclear region, the middle piece and the terminally tufted tail. Nevertheless it is only 46-50 µm in length and the head, including a slightly tight helical nuclear region, is similar in length to the tail. In all macrobiotid species here examined, including those from literature, the spermatozoa within the was deferens always appear folded, with the hinge located between the end of the head and the beginning of the middle piece, thus resembling a long nutcracker. The use of spermatozoan characters as phylogenetic information in tardigrades is also discussed.

2011 - Ultraviolet radiation tolerance in hydrated and desiccated eutardigrades. [Articolo su rivista]
Altiero, Tiziana; Guidetti, Roberto; V., Caselli; Cesari, Michele; Rebecchi, Lorena

So far, most studies on tardigrade resistance to extreme stresses have focused on their responses to several chemical and physical extremes, but there is still a paucity of data regarding the tardigrade responses to ultraviolet radiation. Considering also the future perspectives offered by space flights, we compared the biological responses of two eutardigrade species (Paramacrobiotus richtersi and Ramazzottius oberhaeuseri) to UV irradiation, alone or in combination with multiple stressors (temperature and air relative humidity) in two different physiological conditions (desiccated and hydrated states). Tardigrades were exposed to seven different UV doses (from 10.32 up to 87.72 kJ m-2) in a controlled climatic chamber. The results showed that active and anhydrobiotic tardigrades were able to withstand high doses of ultraviolet radiations. The survival rate of hydrated or desiccated specimens of both species was inversely related to the UV doses, with P. richtersi that better tolerated the increase of UV dose than R. oberhaeuseri. Surprising, the tolerance to physical and chemical extreme stresses is not an exclusive property of desiccated tardigrades; in certain environmental conditions (high air humidity level or low temperature) desiccated tardigrades have a lower or similar withstanding to UV irradiation than hydrated ones. This represents a further demonstration of the uniqueness of this animal group in tolerating extreme stresses. In addition, we demonstrated that high temperatures have a strong impact on tardigrade survival during UV exposition both in hydrated and desiccated animals.

2010 - Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi [Articolo su rivista]
A. M., Rizzo; M., Negroni; Altiero, Tiziana; G., Montorfano; P., Corsetto; P., Berselli; B., Berra; Guidetti, Roberto; Rebecchi, Lorena

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. The glutathione peroxidase was induced by desiccation together with glutathione content. The percentage of fatty acid composition of polyunsaturated fatty acids and the content of thiobarbituric acid reactive substances is higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that possession of antioxidant metabolism could represent a crucial strategy to avoid damages in anhydrobiotic tardigrades.

2010 - Experiments and Models for physics learning in primary school [Relazione in Atti di Convegno]
Mariani, Cristina; Corni, Federico; Altiero, Tiziana; Bortolotti, Carlo Augusto; Giliberti, Enrico; Landi, Laura; Marchetti, Mauro; A., Martini

The project “Little scientists in the lab: Experiments & Models for science learning in primary school”, funded by the Ministry of Education and being currently under development in Italy, is addressed to primary school teachers of the Faculty of Science Education and consequently to pupils. This project proposes a “Model-centered Learning Environment” to build pilot activities for teaching and learning science and physics, based on experimental and modeling activities. The approach for teacher training is to assign group tasks that promote learning.We set up a website to support teachers’ school activities to facilitate and promote communication and exchange of materials between teachers and researchers of the University of Modena and Reggio Emilia, as well as between the teachers themselves. In this paper, we illustrate the general features of the project and focus on preliminary results of an in-service training for teachers in fluids and electricity.

2010 - Hatching phenology and resting eggs in tardigrades [Articolo su rivista]
Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

Little is known about egg dormancy in tardigrades, except for their ability to survive desiccated for a long time. Our previous analyses of life history traits of a reared strain of the leaf litter-dwelling eutardigrade Paramacrobiotus richtersi revealed variation in hatching phenology, suggesting the presence of diapause (resting) eggs in tardigrades. To study adaptive strategies in an unpredictable environment subject to stochastic variability, such as that colonized by tardigrades, we have analysed the hatching phenology of an apomictic triploid cytotype of P. richtersi. The first lab oviposition of mature females collected in the field in the spring and fall as well as cohorts of eggs laid by females born in the laboratory were used. The eggs of all samples, maintained at the same constant experimental conditions, had a high hatching percentage (from 75 to 93%) but high variability occurred in hatching time. Four patterns were identified. First, subitaneous eggs hatched within 30 - 40 days from oviposition. Second, delayed-hatching eggs hatched gradually over 41 – 62 days. Some eggs did not hatch within 90 days from oviposition when water was maintained in the culture. Within this group, 13% of eggs (diapause resting eggs; third category) do not hatch until they are subjected to desiccation followed by rehydration, while 87% never complete their development (abortive eggs; fourth category). The four categories of eggs had no morphological differences. The high variability in the hatching time of tardigrade eggs might be considered a form of bet-hedging.

2010 - I Tardigradi, questi sconosciuti … e questi fenomeni. [Articolo su rivista]
Altiero, Tiziana; Bertolani, Roberto

Vengono descritte le caratteristiche morfologiche e biologiche di questo gruppo di microinvertebrati, valutando le sue relazioni con gli Artropodi ed approfondendo in particolare le capacità di resistenza agli stress degli ambienti estremi. Vengono infine prese in considerazione le potenzialità di questi animali nella ricerca applicata.

2010 - I tardigradi, gli ambienti estremi e i viaggi nello spazio [Articolo su rivista]
Rebecchi, Lorena; Guidetti, Roberto; Altiero, Tiziana

In questo lavoro viene presentata la biologia dei tardigradi e le motivazioni che rendono interessante il loro studio nell’ambito della biologia evoluzionistica. Il primo motivo di interesse è rappresentato dalla molto probabile condivisione del percorso evolutivo seguito dagli artropodi nella sua prima parte. Il secondo motivo è legato alle molteplici strategie riproduttive, mentre il terzo è rappresentato dalla dormienza (diapausa e criptobiosi), sempre presente nei tardigradi terrestri che di fatto, essendo attivi soltanto quando è presente l’acqua, mantengono le caratteristiche di organismi acquatici anche sulle terre emerse. L’ultimo motivo di interesse è rappresentato dalla sorprendente resistenza agli stress chimico-fisici estremi che li rendono un ottimo modello negli studi di biologia spaziale. I tardigradi sono stati infatti selezionati come modello di organismi pluricellulari da utilizzare per esperimenti nello spazio. Nel settembre 2007 sono stati esposti all’ambiente spaziale durante il volo della navicella russa FOTON-M3 che li ha mantenuti nello spazio (“Low Earth Orbit”) per dodici giorni, mentre nel maggio 2011 sono stati ospitati sulla Stazione Spaziale Internazionale (ISS) per sedici giorni grazie all’ultimo volo dello shuttle Endeavour.

2010 - Il progetto per la diffusione della cultura scientifica ‘Piccoli scienziati in laboratorio: Esperimenti & Modelli per la formazione scientifica nella scuola primaria' [Relazione in Atti di Convegno]
Corni, Federico; Giliberti, Enrico; Mariani, Cristina; Altiero, Tiziana; Marchetti, Mauro; A., Martini

Il progetto “Esperimenti & Modelli per la formazione scientifica nella scuola primaria” promosso dal Dipartimento di Fisica e dalla Facoltà di Scienze della Formazione dell’Università di Modena e Reggio Emilia, a cui prendono parte docenti dell’Ateneo provenienti da diversi ambiti disciplinari (matematica, fisica, biologia, scienze della terra e pedagogia), si propone di realizzare attività pilota per la predisposizione di materiali per l’insegnamento e l’apprendimento delle scienze basati sull’impiego dei modelli, denominato Model–centred Learning Environment. L’impatto dell’iniziativa si articola sia a livello locale (formazione iniziale degli insegnanti del Corso di Laurea in Scienze della Formazione Primaria presso la Facoltà di Scienze della Formazione dell’Università di Modena e Reggio Emilia; formazione in servizio e aggiornamento degli insegnanti) che a livello multiregionale (formazione in servizio e aggiornamento). Il coinvolgimento di istituzioni scolastiche e di reti di istituzioni scolastiche in regioni diverse ha lo scopo di verificare le condizioni di replicabilità del modello e di favorirne la diffusione a livello nazionale.In questo contributo verrà presentata la struttura del progetto e i materiali attualmente progettati e realizzati.

2010 - Introduzione elementare all’energia: un laboratorio di scienze per insegnanti di scuola primaria [Relazione in Atti di Convegno]
Altiero, Tiziana; Bortolotti, Carlo Augusto; Corni, Federico; Giliberti, Enrico; R., Greco; Marchetti, Mauro; Mariani, Cristina

L’insegnamento delle Scienze nella Scuola Primaria deve fornire agli alunni le basi per un approccio scientifico ai fenomeni e ancor prima di proporre temi di discipline separate è opportuno che favorisca la costituzione di un insieme di concetti elementari di fondo comuni e trasversali alle discipline stesse. In ciò gioca un ruolo fondamentale la formazione iniziale degli insegnanti che, anche in considerazione delle linee che si evincono dalle bozze della imminente riforma del Corso di Laurea in Scienze della Formazione Primaria, devono poter usufruire di validi supporti disciplinari, senza però dimenticare la profonda interconnessione delle discipline scientifiche nell’interpretazione dei fenomeni quotidiani e dei processi naturali compresi nel curricolo.Nell’anno accademico 2008-2009 è stato proposto nella Facoltà di Scienze della Formazione un Laboratorio di 16 ore sul tema dell’energia a cui hanno collaborato quattro docenti di diverse discipline scientifiche (Fisica, Chimica, Biologia e Scienze della Terra) e un docente di ambito pedagogico.Il tema dell’energia è stato scelto per il suo carattere di trasversalità alle discipline scientifiche e come contesto paradigmatico in cui costruire un linguaggio e un approccio comune. Il modello energetico adottato si ispira a quello del Karlsruhe Physics Course [1] in cui l’energia è definita a partire dalle sue proprietà di conservazione e introdotta come ente regolatore dei processi naturali. In questo senso viene superata l’idea di “trasformazione dell’energia” introducendo il concetto di “trasferimento di energia” fra portatori, e di esistenza di diverse “forme di energia” pensando piuttosto l’energia come entità sempre associata a un portatore.Il laboratorio è stato aperto con una introduzione sul modello dei portatori e dei trasferitori di energia e si è poi sviluppato con diverse attività di interpretazione da parte degli studenti di situazioni sperimentali riconducibili alle quattro discipline coinvolte. Le attività sperimentali e di interpretazione sono state svolte direttamente dagli studenti, guidati da alcune schede-guida progettare dai singoli docenti e omogenee per tutto il percorso. Prima e dopo l’intervento gli studenti hanno compilato, in modo anonimo, un test di ingresso e un test di uscita.In questo lavoro, dopo una breve introduzione sull’approccio all’energia scelto, verranno presentate in dettaglio la struttura dell’attività del laboratorio e le principali conclusioni che si possono trarre dall’analisi dei materiali prodotti dagli studenti.

2009 - Survival and DNA degradation in anhydrobiotic tardigrades [Articolo su rivista]
Rebecchi, Lorena; Cesari, Michele; Altiero, Tiziana; A., Frigieri; Guidetti, Roberto

Anhydrobiosis is a highly stable state of suspended animation in an organism due to its desiccation, which is followed by recovery after rehydration. Changes occurring during drying could damage molecules, including DNA. Using as a model organism the anhydrobiotic tardigrade Paramacrobiotus richtersi, we have evaluated the effects of environmental factors, such as temperature and air humidity level (RH), on the survival of desiccated animals and on the degradation of their DNA. Tardigrades naturally desiccated in leaf litter and tardigrades experimentally desiccated on blot paper were considered. Replicates were kept at 37°C and at different levels of RH for 21 days. RH values and temperature, as well as time of exposure to these environmental factors, have a negative effect on tardigrade survival and on the time required by animals to recover active life after desiccation. DNA damages (revealed as single strand breaks) occurred only in desiccated tardigrades kept for a long time at high RH values. These results indicate that during the anhydrobiotic state, damages take place and accumulate with time. Two hypotheses can be formulated to explain the results: i. oxidative damages occur in desiccated specimens of P. richtersi, ii. high temperature and high RH values change the state of the disaccharide trehalose, reducing its protective role.

2009 - Tardigrada (Water Bears) [Capitolo/Saggio]
Bertolani, Roberto; Altiero, Tiziana; D. R., Nelson

Encyclopedia of Inland Waters is a new 3-volume reference work, pulling together all the key information in one source from the leading publisher in the field. Inland aquatic habitats occur worldwide at all scales from marshes, swamps and temporary puddles, to ponds, lakes and inland seas and from streams and creeks to rolling rivers. Vital for biological diversity and ecosystem function and as resources for human life, commerce and leisure, inland waters are a vital component of life on Earth. The Encyclopedia of Inland Waters describes and explains all the basic features of the subject, from water chemistry and physics, to the biology of aquatic creatures and the complex function and balance of aquatic ecosystems of varying size and complexity. In the chapter here reported, tardigrades are considered in their various aspects: morphology and anatomy, classification, phylogeny and evolution, reproduction and development, ecology, zoogeography, methods. The chapter is illustrated with many original figures.

2009 - Tardigrade resistance to space effects: first results of the experiment of LIFE – TARSE mission on FOTON-M3 (September 2007) [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; Guidetti, Roberto; Cesari, Michele; Bertolani, Roberto; M., Negroni; M. A., Rizzo

The TARSE (TArdigrade Resistance to Space Effects) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Space flight induced an increase of glutathione content and its related enzymatic activities. Catalase and SOD decreased with space flight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades moulted and females laid eggs. Several eggs hatched and the newborns exhibited normal morphology and behavior.

2008 - A new discovery of Novechiniscus armadilloides (Schuster, 1975) (Tardigrada, Echiniscidae) from Utah, USA with considerations on non-marine Heterotardigrada phylogeny and biogeography. [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; E., Jacobsen; Bertolani, Roberto; R. M., Kristensen

The discovery of a new population of the non-marine heterotardigrade Novechiniscus armadilloides from Utah, USA, allowed us to re-analyze the species by means of scanning electron microscopy and differential interference contrast microscopy. This analysis confirmed the presence of bar-shaped, unpaired segmental plates and of a long filament E, other than the filament A always present in the class Heterotardigrada. It also provided additional information on characters not explicitly cited in the previous descriptions of this monotypic genus, such as details of the head cirri and clavae, details of the buccal tube and of the pharyngeal bulb, sculpture of the dorso-lateral and leg plates, details of the claws. The population is bisexual, but no secondary sexual dimorphism was observed. The male and female gonopore were described. New characters such as red eyes and red body color were used in analyzing the phylogeny of the family Echiniscidae. The phylogeny and biogeography of non-marine heterotardigrades provide intriguing questions for further research.

2008 - Diapause in tardigrades: a study of factors involved in encystment [Articolo su rivista]
Guidetti, Roberto; D., Boschini; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

Stressful environmental conditions limit survival, growth, and reproduction, or these conditions induce resting stages indicated as dormancy. Tardigrades represent one of the few animal phyla able to perform both forms of dormancy: quiescence and diapause. Different forms of cryptobiosis (quiescence) are widespread and studied, while little attention has been devoted to adaptive meaning of the encystment (diapause). Our goal was to determine environmental factors and token stimuli involved in the encystment process of tardigrades. The eutardigrade, Amphibolus volubilis, a species able to produce two types of cysts (type 1 and type 2), has been considered. Laboratory experiments and long-term study on cyst dynamics of a natural population were conducted. Laboratory experiments demonstrate that active tardigrades collected in April produced mainly type 2 cysts, whereas animals collected in November produced mainly type 1 cysts, indicating that the different responses are functions of the physiological state at the time they were collected. Dynamics of the two types of cyst show opposite seasonal trends: type 2 cysts are present only during the warm season and type 1 cysts, present during the cold season. Temperature represents the environmental factor involved in induction, maintenance, and termination of the cyst. We also evidenced that A. volubilis is able to perform both diapause and cryptobiosis, even overlapping the two phenomena. The induction phase of tardigrade encystment can be compared to the induction phase of insect diapause, also indicating an involvement of endogenous factors in tardigrade encystment. As in insect diapause, tardigrade encystment can be considered a diapausing state controlled by exogenous and endogenous stimuli.

2008 - Hsp levels and DNA integrity in anhydrobiotic tardigrades [Abstract in Atti di Convegno]
Guidetti, Roberto; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

Dehydrating cells can lead to massive damage in molecular organization, often resulting in cell death and, consequently, death of the organism. In anhydrobionts, several bioprotectants, e.g. sugars and stress proteins, play a role in avoiding these damages. In tardigrades (micrometazoans able to perform anhydrobiosis in any stage of their life cycle), bioprotectants are certainly involved, but their nature and role is only understood in part. We evaluated the Hsp70 and Hsp90 levels in hydrated and desiccated animals of two eutardigrade species: Macrobiotus richtersi and Amphibolus volubilis. For both species, we found no significant differences in the level of both Hsps between hydrated and anhydrobiotic specimens.Also, DNA can be damaged by dehydration. In several unicellular organisms, even though characterized by desiccation tolerance, dramatic DNA damages can occur during desiccation or in the dry state that follows. It has been hypothesized that DNA damages could also occur in anhydrobiotic tardigrades. To verify this hypothesis, we evaluated tardigrade survival and DNA desiccation-induced double (DSBs) and single (SSBs) strand breaks in specimens of M. richtersi naturally dried within leaf litter as well as experimentally desiccated in our lab under controlled conditions. Dry specimens were also exposed to thermal stress (37°C) at different values of air humidity (RH). An inverse relationship between M. richtersi survival and RH levels was found. Moreover, tardigrades dried within leaf litter, when re-hydrated had a faster recovery of active movement than those dehydrated on paper. No visible DNA DSBs were observed, but preliminary data on SSBs seems to bring different results, which need to be confirmed.

2008 - TArdigrade Resistance to Space Effects (TARSE): primi risultati della missione LIFE su Foton-M3 [Abstract in Atti di Convegno]
Altiero, Tiziana; Guidetti, Roberto; Bertolani, Roberto; A. M., Rizzo; Rebecchi, Lorena

Il progetto TARSE, parte della linea ASSC del progetto MoMa dell’ASI ha proposto i tardigradi come modello da utilizzare in esperimenti nello spazio. Gli obiettivi specifici erano: verificare gli effetti dell’ambiente spaziale su sopravvivenza, cicli vitali, induzione di bioprotettori, danni al DNA dovuti a radiazioni o a stress da essiccamento. Sono stati condotti quattro esperimenti sull’eutardigrado Macrobiotus richtersi, con animali in stato essiccato (all’interno di un microcosmo o su carta) ed idratato (alimentati o a digiuno). Gli animali hanno volato per 12 giorni nella navicella spaziale Foton M3, utilizzando la facility Biokon (Kayser Italia). Le attività post-volo previste consistevano in analisi: della sopravvivenza, dei parametri del ciclo vitale, biochimiche e molecolari. Erano previsti, e in parte sono stati realizzati, esperimenti di controllo a terra, simulando la temperatura del volo, o la microgravità (con RPM). I tassi di sopravvivenza, simili a quelli degli esperimenti a terra, risultavano elevati per gli individui essiccati sia nel microcosmo 94,4±4,8%) che su carta (78,9±25,2%) e per gli animali idratati digiuni (60,6±8,3%). Simili erano anche le quantità di Hsp 70 riscontrate negli esperimenti a terra.Non sono stati evidenziati danni alla doppia elica di DNA. Durante il volo, le femmine attive hanno deposto uova, schiuse in gran numero e in tempi più brevi del controllo a terra; i giovani nati mostravano normale morfologia e comportamento.

2008 - Tardigrade resistance to space effects (tarse): first results of the mission life on foton-m3 [Abstract in Atti di Convegno]
Rebecchi, Lorena; Guidetti, Roberto; Altiero, Tiziana; Bertolani, Roberto; A. M., Rizzo

Anhydrobiosis is a highly stable and reversible state of suspended life due to complete organism desiccation. Anhydrobiotic tardigrades show extraordinary resistance to physical and chemical extremes, far exceeding tolerance ranges of active animals. Consistent with these potentialities, the Mission LIFE on Foton M3 - TARSE (TArdigrade Resistance to Space Effects), part of the line ASSC of the ASI MoMa project, aimed to analyse the effects of space environment on this animal model. Four experiments have been carried out using Macrobiotus richtersi. In September 2007 desiccated (in microcosm or on paper) and hydrated tardigrades (fed or starved) have flown on Foton M3 for 12 days, using the facility Biokon (by Kayser Italia). As control, ground experiments miming the flight temperatures are currently running. To date we have complete results about survival and preliminary data on life cycle parameters. The highest survival rate has been obtained for desiccated specimens in microcosm (94.4±4.8%) and on paper (78.9±25.2%), and for hydrated starved animals (60.6±8.3%). The lowest survival rate has been recorded for hydrated fed animals (6.8±9.9%). During the flight: i) active females laid eggs, ii) several eggs hatched, iii) the hatching time was very short, and iv) newborns exhibited normal morphology and behaviour. After the flight, adults laid eggs and the reared newborns reached sexual maturity and laid eggs too. Currently we are analysing: i) genomic DNA quality, ii) Hsp expression, iii) oxidative stress. Studies on genomic DNA quality and oxidative stress will allow us to verify possible radiation effects, while Hsp expression will permit to investigate the role of this bioprotectant. Finally, ground experiments on RPM are settled to verify the eventual effects of microgravity.

2007 - Anhydrobiosis: the extreme limit of desiccation tolerance [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; Guidetti, Roberto

Extreme habitats give rise to strong stressors that lead organisms to die or to possess specific adaptations to those stressors. One of the most widespread adaptations is quiescence, a common term for several strategies, including anhydrobiosis, a highly stable state of suspended animation due to complete desiccation pending recovery by rehydration. Anhydrobiosis is widespread in nature in a wide taxonomic variety among bacteria, protists, metazoans and plants. Using as model organisms, mainly tardigrades, micrometazoans able to enter anhydrobiosis in any phase of their life cycle from egg to adult, this review presents the response to desiccation from molecules to cells and organisms. Particular emphasis has been done with studies devoted to elucidate phenomena such as the long-term resistance in a desiccated state, the extraordinary resistance to chemical and physical extremes, the morphological, physiological, biochemical, and molecular constraints allowing organisms to enter and to survive anhydrobiosis, and the evolutionary meaning of life without water.

2007 - Approccio molecolare, modalità riproduttive e specie criptiche nei tardigradi. [Abstract in Atti di Convegno]
Bertolani, Roberto; Guidetti, Roberto; Cesari, Michele; Altiero, Tiziana; Rebecchi, Lorena

Gli studi molecolari hanno portato ad attribuire i tardigradi agli Ecdysozoa e confermato sia la loro appartenenza ai Panarthropoda, che la loro suddivisione in ordini. Ancora assai limitato è invece l’utilizzo della biologia molecolare per discriminare le loro specie. Numerosi sono, infatti, i ritrovamenti di tardigradi attribuibili alla medesima morfospecie, ma distinti in popolazioni unisessuate e bisessuate, spesso diverse anche per grado di ploidia. Le indagini condotte finora non hanno però consentito di stabilire con certezza se tra queste popolazioni vi fosse o meno flusso genico. Utilizzando come marcatore una sequenza di 640 bp del gene mitocondriale COI, sono state confrontate popolazioni riferibili all’eutardigrado Macrobiotus richtersi con diversa provenienza geografica (Modena e Trento) e verificate le loro modalità riproduttive. In entrambe le province sono state rinvenute, in località distinte, sia popolazioni anfimittiche (diploidi) che partenogenetiche telitoche (triploidi e ameiotiche). L’analisi delle sequenze ha rilevato notevole somiglianza nell’aplotipo delle popolazioni partenogenetiche, pur se geograficamente distanti, e forti differenze tra queste e le anfimittiche. Queste sono coerenti con quanto da noi già riscontrato tra popolazioni anfimittiche e partenogenetiche di un’altra morfospecie (Richtersius coronifer) utilizzando allozimi. La costanza e l’ampiezza delle differenze nell’aplotipo evidenziano che non c’è flusso genico tra le popolazioni e che siamo in presenza di specie criptiche. Data l’esistenza di numerose morfospecie di tardigradi con più modalità riproduttive, il loro studio a livello molecolare, associato a quello della loro distribuzione geografica, dovrebbero portare nuova luce sulla microevoluzione all’interno del phylum e, più in generale, sui microinvertebrati caratterizzati da forte dispersione passiva.

2007 - Energy allocation in two species of Eutardigrada [Articolo su rivista]
Guidetti, Roberto; C., Colavita; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

To improve our knowledge on life histories in tardigrades and the energy allocated for their reproduction and growth, we have studied two species (Macrobiotus richtersi and Hypsibius convergens) differing in evolutionary histories, diet and ways of oviposition. For both species we considered a bisexual population dwelling in the same substrate. In both species we investigated energy allocations in males with a testis rich in spermatozoa and females with an ovary containing oocytes in advanced vitellogenesis. The age of the specimens were estimated on the basis of buccal tube length and body size and the body and gonad areas were calculated using an image analysis program. In both species females reach a larger size than males. Macrobiotus richtersi has significantly longer buccal tube and wider body area than H. convergens. Statistical analyses show that buccal tube has a positive correlation with body area and gonad area. For an estimate of the relative energy allocated for reproduction in one reproductive event (relative reproductive effort = RRE), we have used the ratio between gonad area and body area. In males of both species, the absolute amount of energy and the RRE is statistically lower than that of females. Males and females of H. convergens have a RRE higher than those of M. richtersi. In M. richtersi, the gonad increases proportionally more when animals are large (old), whereas in H. convergens this direct relationship is not detectable. In M. richtersi the energy allocated for a reproductive event increases during the life of the females. In males, the increase of the gonad size is progressive during the animal life. In each reproductive event, females of H. convergens allocate a lower amount of energy in absolute value when compared to M. richtersi. Nevertheless, when considering the RRE, their investment is higher than that of M. richtersi.

2007 - Indagini sulla sopravvivenza ad essiccamento e stress termico e sulla ripresa della vita attiva in tardigradi colonizzanti ambienti a variabilità stocastica [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Cesari, Michele; A., Frigieri; V., Lencioni; Bertolani, Roberto; Guidetti, Roberto

La possibilità di attuare la criptobiosi consente ai tardigradi di colonizzare ambienti estremi (deserti, montagne elevate, o regioni polari) preclusi alla maggior parte degli altri organismi e, nelle zone temperate, di vivere in habitat particolari caratterizzati dall’alternanza stocastica di periodi di siccità e pioggia, o di congelamento e scongelamento. Studi sulla criptobiosi nei tardigradi sono stati rivolti ad individuare le molecole bioprotettrici, soprattutto il trealosio, prodotte quando gli animali entrano in questo stato. Nostre indagini, basate sull’interazione tra osservazione in natura e sperimentazione in laboratorio, hanno riguardato l’effettiva capacità di sopravvivenza dei tardigradi in anidrobiosi e i relativi aspetti microevolutivi. Esse hanno portato all’individuazione di differenze nel tasso di sopravvivenza, sia tra specie, che tra popolazioni della stessa specie provenienti da aree geografiche diverse. Abbiamo ora inteso considerare gli effetti combinati di stress da essiccamento e da calore. Sono stati pertanto condotti alcuni esperimenti utilizzando Macrobiotus richtersi, eutardigrado facilmente reperibile nella lettiera di bosco sottoposta a ripetuti e imprevedibili eventi di disidratazione e reidratazione (oltre che a congelamento e scongelamento). Esemplari lasciati seccare all’interno di un campione di lettiera di bosco raccolto in natura e quindi ulteriormente stressati mantenendoli a 37°C per 21 giorni, non evidenziano diminuzioni significative della sopravvivenza dopo la reidratazione. Tuttavia, il tempo necessario per riprendere la vita attiva da parte di questi animali non resta uguale, ma aumenta in relazione diretta al tempo trascorso a 37°C. Diversamente, esemplari seccati in un campione di lettiera di bosco e stressati a 37°C per 21 giorni, ma mantenuti a diversi livelli di umidità relativa dell’aria (RH, 80%, 25%, circa 0%), mostrano, dopo reidratazione, una sopravvivenza inversamente proporzionale al valore di RH. Inoltre, il tempo necessario per la ripresa della vita attiva è direttamente proporzionale al valore di RH a cui gli animali stessi sono stati esposti. Un risultato simile (ma con sopravvivenza complessivamente minore) è emerso da animali essiccati sperimentalmente su carta a condizioni controllate e quindi sottoposti a 21 giorni di stress termico, sempre a 37°C. Alcuni autori sostengono che la criptobiosi, o almeno l’anidrobiosi, comporti un danno del DNA e quindi che la ripresa della vita attiva sia accompagnata da fenomeni riparativi del DNA stesso. Questo si accorderebbe con il maggior tempo necessario agli animali sottoposti a forti livelli di stress per riprendersi. Tuttavia, nostre indagini sul DNA estratto da esemplari di M. richtersi essiccati su carta e stressati a 37°C non hanno verificato la presenza di rotture dell’acido nucleico, indicando che la causa del ritardo nella ripresa della vita attiva deve essere ricercata in altri tipi di molecole.

2007 - The mission Foton-M3 t.o evaluate the tardigrade resistance to space effects [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Guidetti, Roberto; Bertolani, Roberto; A. M., Rizzo

Tardigrades are microscopic animals able to pass over dehydration stress entering anhydrobiosis (i.e. dramatically reducing their metabolism) in any stage of their life. Using this strategy, tardigrades survive to extremely warm and dry environments excluded to most animals. Anhydrobiosis also gives rise to resistance against other stresses (i.e. temperatures over 100°C, freezing and ionizing rays). Trehalose is involved in anhydrobiosis playing an important role to protect cells. This sugar is currently used as additive for protecting mammalian cells during cryopreservation. Other molecules (e.g. heat shock proteins, Hsps) seem to be further keys to understand the anhydrobiotic mechanisms.The study here proposed (TARSE: Tardigrade Resistance to Space Effects) is part of the line ASSC (Animal Space Stress and Countermeasures) of the ASI MoMa project. Its aim is to study the ability of tardigrades to survive to space environments using as a tool anhydrobiotic and hydrated specimens of Macrobiotus richtersi, in light to lead to a development of molecules or mechanisms required to stabilize cells. Animals will fly using the facility Biokon (by Kayser Italia) during the mission Foton-M3. As a ground control, identical replicates will be maintained in Modena laboratory. Particular emphasis will be placed on the resistance to desiccation and to radiation stresses. The specific goals are: i) to verify the effect of space environment on survival of both desiccated and hydrated tardigrades, ii) to detect bioprotectant (e.g. Hsps) induction, iii) to verify the eventual damages on DNA due either to radiation or desiccation stresses, and the ability to repair them.

2006 - A new species of freshwater tardigrades from Disko Island (Greenland) increases an unsolved paradox in tardigrade systematics [Articolo su rivista]
Guidetti, Roberto; Altiero, Tiziana; Jg, Hansen

During the Workshop on Arctic tardigrades at the Danish Arctic Station (Qeqertarsuaq, Disko Island, Greenland) an undescribed species of Dactylobiotus was found in freshwater sediments of the Isunngua spring. We have the honour and pleasure to describe this new taxon that we dedicate to all participants of that symposium, naming the species Dactylobiotus octavi sp. n. The animals appear similar to Dactylobiotus dispar and Dactylobiotus haplonyx with the presence of a very short secondary branch in the claws of the first three pairs of legs, but they differ from these species in the size of claw and buccal tube width. This new species also has peculiar ornamented eggs. The eggshell consists of bowl-like processes, each one surrounded by a band of fine pores. A morphological parsimony analysis to identify phylogenetic relationships among D. octavi sp. n. and the other Dactylobiotus species was performed, obtaining inconclusive results. The discovery of this new species increases an unsolved paradox in tardigrade systematics related to the presence of closely related species which share a very similar morphology of the animals but clearly differ in their egg morphology, while, conversely, there are species belonging to different evolutionary lines that have similar eggs, but very different adult morphology. The finding of D. octavi sp. n. increases the already high number of species found in Disko Island and once again underlines the importance of tardigrades in the biodiversity of the Arctic area.

2006 - Dynamics of long-term anhydrobiotic survival of lichen-dwelling tardigrades [Articolo su rivista]
Rebecchi, Lorena; Guidetti, Roberto; S., Borsari; Altiero, Tiziana; Bertolani, Roberto

It is not rare to find in references that anhydrobiotic tardigrades can survive for more than a century. However, a closer look at the empirical evidence provides very little support that tardigrades are capable of surviving dried for such a long time. In order to resolve this discrepancy, we carried out a study to evaluate the long-term survival of naturally dried tardigrades. A large fragment of dry lichen (Xanthoria parietina) was collected in the field two days after a rainy day in 1999. The dry lichen was stored inside a paper bag in the laboratory at room temperature and humidity and under atmospheric oxygen exposure. Replicates of the dry lichen were re-hydrated after various time periods of storage, with all tardigrades extracted and the survivors enumerated. Five species of tardigrades were found, but two of them only occasionally. Ramazzottius oberhaeuseri, Echiniscus testudo and Echiniscus trisetosus were sufficiently represented for statistical analysis. At the beginning of the experiment 91.1% of R. oberhaeuseri and 71.7% of Echiniscus spp. were alive. R. oberhaeuseri survived up to 1604 days, while Echiniscus spp. lived up to 1085 days. Recovery after four years of anhydrobiosis has to be considered a very good long-term survival, which is important from an ecological and evolutionary point of view.

2006 - Energy allocation in the reproductive events of Macrobiotus richtersi and Hypsibius convergens (Eutardigrada). [Abstract in Atti di Convegno]
Altiero, Tiziana; Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena

The knowledge of life histories in tardigrades is still limited, while an evaluation of the energy allocation for their reproduction has been considered very little. To improve our knowledge on these topics, we have studied two species differing in evolutionary histories, diet and ways of oviposition. Macrobiotus richtersi (Macrobiotidae) is carnivorous and lays “free” ornamented eggs, while Hypsibius convergens (Hypsibiidae) is certainly not carnivorous and lays smooth eggs within the exuvium. For both species we considered a bisexual population dwelling in the same substrate, a beech leaf litter collected on the Apennines (Piane di Mocogno, Modena, Italy) at 1200 m a.s.l. Both species are iteroparous. In M. richtersi, the maturative patterns of male and female gonads follow the respective models proposed by Rebecchi & Bertolani (1994). In H. convergens the male germ cell maturation is continuous and follows the previous male model, whereas the female germ cell maturation does not strictly follow the stages described for M. richtersi and other eutardigrade species.With regard to the energy allocation, males with testis rich in spermatozoa and females with ovary containing oocytes in advanced vitellogenesis have been considered in both species. The age of those specimens has been estimated according to the buccal tube length. Their body and gonad areas have been evaluated with an image analysis program. In both species females reach a larger size than males. Macrobiotus richtersi has statistically longer buccal tube and wider body area than H. convergens. Statistical analysis evidences that the buccal tube length is positively related to the body area and to the gonad area. For an estimate of the relative energy allocated for the reproduction in one reproductive event (here called RRE = relative reproductive effort), we have used the ratio: gonad area/body area. In males of both species, the absolute amount of energy and, above all, the RRE is statistically lower than that of females. In M. richtersi, the RRE of both males and females is directly related to the age of the animal, whereas in H. convergens this direct relationship is not detectable. This means that in M. richtersi the energy allocation for a reproductive event increases during the life of the animal. In each reproductive event, due to their smaller size, in absolute value, females of H. convergens allocate a lower amount of energy with respect to M. richtersi, but if we consider the RRE, their investment results higher than that of M. richtersi.

2006 - Phenotypic variations in the life history of two clones of Macrobiotus richtersi (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Altiero, Tiziana; Rebecchi, Lorena; Bertolani, Roberto

A comparative study of life history traits of two clones (CDMr01 and CDMr02) of a triploid thelytokous apomictic population of the eutardigrade Macrobiotus richtersi has been carried out. Both clones were reared under the same lab conditions: temperature of 14 degrees C, photoperiod of 12 h/12 h (L/D), and nematodes ad libitum as food. Statistical analysis of the life history traits considered has indicated interclonal variability. The two clones were significantly different in the number of eggs per clutch (fertility), number of eggs laid per female per life span (fecundity), hatching percentage of eggs and hatching time. Similarities between clones have been observed with regard to life span, total number of ovipositions per life span, and age at first oviposition. In addition, significant differences in hatching time, with eggs hatched over a long period, were found within each clone. Interclonal variability in life history traits indicated the effects of genetic factors, whereas intraclonal variability reflected the effects of environmental factors. The evolutionary and adaptive significance of the life history phenotypic variations is discussed.

2006 - Risposte in modelli animali agli effetti dell’ambiente spaziale: i tardigradi, organismi resistenti a stress di ambienti estremi. [Abstract in Atti di Convegno]
Rebecchi, Lorena; Boschini, Deborah; Altiero, Tiziana; Bertolani, Roberto; Guidetti, Roberto

I 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-sho

2006 - Tardigrades from Stelvio National Park, Central Alps (Italy) [Abstract in Atti di Convegno]
Guidetti, Roberto; Bertolani, Roberto; B., Maiolini; Altiero, Tiziana

Tardigrades have been collected in different habitats in an Alpine glacial valley (Val de la Mare, Stelvio National Park, Trentino, Italy), between 2200 and 2300 m a.s.l. Submerged mosses of streamlets (2 samples), turf (1 sample) and mosses on rocks (2 samples) have been considered. These habitats hosted a very rich and diversified tardigrade fauna. Particularly rich in tardigrades were the submerged mosses, which comprised 9 species of eutardigrades belonging to 3 different families and 8 genera. Worthy of note are the first record of Borealibius zetlandicus in Italy (a species with a boreo-alpine geographic distribution) and the presence of two kinds of eggs in Murrayon pullari, a situation reported only once in tardigrades. Within the turf, a poorly known habitat, we found only one species, a Macrobiotus belonging to the “hufelandi group”, certainly new to science. Mosses on rocks contained 8 species (three echiniscid heterotardigrades and 5 belonging to two families of eutardigrades), different from those found in the other two habitats; three of them (two Ramazzottius and one Hypsibius) are probably new to science. In total, 18 species have been identified, four of which are new to science, one new to Italy and three new to the Trentino province. These results confirm the biogeographic significance of many tardigrades and suggest the need for more specific studies on tardigrade distribution focused on high altitudes and glacial areas of the temperate zones, which should be considered as biogeographic islands.

2005 - Maintenance of variability in tardigrades: parthenogens vs amphimictics in Richtersius coronifer [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; V., Rossi

Parthenogenetic and amphimictic populations within a same morphospecies are frequently reported in non-marine tardigrades. A same morphospecies can have diploid bisexual population(s) reproducing by amphimixis, and polyploid (rarely diploid) unisexual population(s) carrying out apomixis. We assess the breeding system of two Italian populations and one Swedish population of the eutardigrade Richtersius coronifer evaluating sex-ratios, analyzing karyotypes and examining genetic structure using allozyme markers. The populations differed in sex ratio and reproductive modes. One Italian population was bisexual and amphimictic, while the other two were unisexual and parthenogenetic. Data from several years show no evidence of cyclical parthenogenesis. All populations were diploid with the chromosome number (2n = 12) and had bivalents in the oocytes. The chromosome pairing found in the parthenogen oocytes is evidence of a meiotic maturation. The presence of chiasmata in oocytes suggests that recombination exists, even though we do not know how the diploid chromosome number is restored. With regard to the genetic structure, we analyzed 14 loci both in the amphimictic and in the parthenogenetic populations. Only three loci (GPI, IDH and PGM) were polymorphic. The amphimictic population had a degree of genetic variability (mean heterozygosity higher than 0.25) higher than that of the parthenogenetic population. In fact, in all female populations, allele frequencies at all 3 loci deviated from Hardy-Weinberg expectations due to lack of heterozygosity, in accordance with the cytological results evidencing an automictic parthenogenesis. GPI locus was represented by 6 alleles in the amphimictic populations and only by one in the automictic populations, but IDH and PGM were represented by 4 alleles both in the amphimictic and in the automictic populations, evidencing a maintained (or acquired) variability. We cannot exclude that the recent derivation of the asexual populations from few lineages (demographic bottlenecks) could explain the loss of genetic variation, in terms of number of alleles per locus, even though in populations affected by recent demographic bottleneck we expect to observe a relative heterozygosity excess due to the fact that allelic diversity decreases more rapidly than heterozygosity. We found high genetic differentiation between populations of R. coronifer. The observed value of Fst (0.281, i.e. 28% of the total genetic variation is due to differences between populations) is above the threshold (0.25) usually considered indicative of high differentiation among populations. Genetic divergence is easy to explain in this species: differences in reproductive mode, fragmented habitat, patchy distribution and the ensuing difficulty in crossing from one moss fragment to another (i.e. low gene flow and high likelihood of population bottlenecks) are probably all acting on R. coronifer populations.

F., Baraldi; A., Severino; C., Bellavere; A., Gandolfi; Altiero, Tiziana; Rebecchi, Lorena; V., Rossi

We analysed the genetic structure of three populations of Heterocypris from temporary ponds on the island of Lampedusa (Italy) where both unisexual (all females) and bisexual populations were found. Population from Cavallo Bianco is made up of females only with typical morphological feature of H. incongruens. In the bisexual population from Aria Rossa adult females present a lobe-like expansion (lamella hyalina) on the posterior margin of the left valve. This is a typical morphological feature of H. barbara. In bisexual population from Vallone della Forbice both female morphotypes are syntopic. Isoenzymatic analysis shows that adult females without lamella hyalina are apomictic, whereas females with this feature are amphimictic.To evaluate the taxonomic status and the phylogenetic relationships in Heterocypris complex from Lampedusa, we used sequences of the mtDNA 16 S ribosomal subunit and sequences of the Elongation factor-1 nuclear gene. In addition, karyotype analysis was performed with Giemsa, fluorocromes and C-banding. For comparison, we analysed a clonal lineage from a parthenogenetic population from a northern Italian rice-field, individuals from a Spanish amphimictic population of H. incongruens (temporary pond of Laguna Espolla) and individuals from a Spanish bisexual population of H. barbara.Heterocypris populations from Lampedusa can be grouped into two main groups: a cluster including parthenogenetic females without lamella from Cavallo Bianco (20 chromosomes two of which unpaired), the parthenogenetic lineage of H. incongruens from northern Italy and H. incongruens from bisexual population from Spain (female 2n = 10A+10X; male 2n = 10A+5X). A second cluster including amphimictic females with lamella from Aria Rossa (female 2n = 10A+10X; male 2n = 10A+5X), parthenogenetic females without lamella and amphimictic females with lamella from Vallone della Forbice and H. barbara bisexual population from Spain. This result confirms that two species of Heterocypris coexist on Lampedusa Island: H. incongruens and H. barbara. Heterocypris incongruens parthenogenetic females from Cavallo Bianco show the typical morphological feature of the species. Within the H. barbara group, two female lineages are distinguishable according to their morphotypes and reproductive modes: amphimictic females with lamella and parthenogenetic females without lamella. These lineages are syntopic in Vallone della Forbice.Our data support the hypothesis of inter-specific hybridisation between coexisting syngamic and clonal lineages of Heterocypris. The close taxonomic association of asexual and sexual females within H. barbara cluster imply a recent origin for asexual lineages.

Altiero, Tiziana; Rebecchi, Lorena

Organisms can experience conditions that are limiting for growth, survival or reproduction in changing environments. The form and nature of this variability differ among habitats and this led to the evolution of different life histories. A comparative study of some life history traits has been realized under experimental conditions considering lineages of two eutardigrade species: Macrobiotus richtersi (Macrobiotidae) and Hypsibius dujardini (Hypsibiidae). These two species have different habitats, food preferences, reproductive mode, dormancy traits and phylogenetic position. Macrobiotus richtersi is very frequent in leaf litter, feeds on micrometazoans and is able to carry out anhydrobiosis. Hypsibius dujardini mainly colonises freshwater and feeds on algae. Our lineage of H. dujardini never carried out anhydrobiosis under experimental conditions. Both lineages are thelytokous, even though M. richtersi (clone CDMr01) is triploid apomictic, while H. dujardini is diploid automictic. Both species are iteroparous. The lineages were reared under the same laboratory conditions: 14°C temperature and photoperiod of 12h/12h (L/D). Macrobiotus richtersi fed on nematodes, H. dujardini fed on unicellular algae of the genus Selenastrum. The following life history traits were considered: lifespan, age at first oviposition, number of clutches per lifespan, number of eggs per clutch (fertility), number of eggs per lifespan (fecundity), hatching time and hatching percentage.Inter- and intraspecific variability have been observed for most life history traits analysed. Mean number of clutches per lifespan was similar in both species. Females of M. richtersi were characterized by a longer lifespan, and a later age at first oviposition than H. dujardini. Fertility and fecundity of M. richtersi were higher than those of H. dujardini. Lastly, H. dujardini had a lower hatching percentage, and a shorter hatching time than those of M. richtersi. In this last species, an unsynchronized hatching phenology and the presence of resting eggs have been observed. Interspecific variability may be explained by the different habitat colonised that have selected different life history traits. Hypsibius dujardini inhabits substrates normally not subject to desiccation, while M. richtersi inhabits substrates periodically subject to desiccation. In this last condition, strategies to overcome transient and unpredictable environmental conditions are selective such as the well-known capability to enter anhydrobiosis, but also the new finding of a long hatching time.

2004 - Experiences with dormancy in tardigrades. [Articolo su rivista]
Bertolani, Roberto; Guidetti, Roberto; K. I., Joensson; Altiero, Tiziana; D., Boschini; Rebecchi, Lorena

Tardigrades often colonise extreme habitats, in which they survive using both types of dormancy: quiescence and diapause. Together with nematodes and bdelloid rotifers, tardigrades are known to enter quiescence (with several forms of cryptobiosis: anhydrobiosis, cryobiosis, anoxybiosis, osmobiosis) at any stage of their life cycle, from egg to adult. Entering anhydrobiosis, tardigrades contract their body into a so-called tun, loosing most of their free and bound water (&gt; 95%), synthesizing cell protectants (e.g., trehalose, glycerol, heat shock proteins) and strongly reducing or suspending their metabolism. Our research on cryptobiosis focused on some ecological and evolutionary aspects. We evaluated: i) the long-term anhydrobiotic survival by comparing quantitative data on recovery from naturally induced desiccation in several species of tardigrades; ii) differences in survival patterns between species and populations by experimentally inducing anhydrobiosis and cryobiosis; iii) phenotypic factors affecting anhydrobiotic survival. As regards diapause, we considered encystment and eggs. Encystment involves at least the synthesis of new cuticular structures. Morphological changes during cyst formation are more complex than those involved in tun formation. We analyzed more in detail encystment processes, comparing a semiterrestrial with a limnic species. Several inter-specific differences have been identified, other than the production of two types of cysts in the semiterrestrial species. Our analysis of life history traits of a laboratory reared strain of a soil tardigrade revealed a particular hatching phenology that involved the production of both subitaneous and resting eggs. The latter need a cue to hatch (dehydration followed by re-hydration). In addition, the evolutionary meaning of dormancy in tardigrades is discussed.

2003 - First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Altiero, Tiziana; Rebecchi, Lorena

Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigrade species, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populations exhibit a diploid chromosome number 2n = 12 and no sex chromosomes were identified. DAPI staining and C-banding data indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at male meiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show no evidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotene stage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many other metazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromosomes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionary significance of achiasmatic meiosis is discussed.

2003 - Reproductive modes and genetic polymorphism in the tardigrade Richtersius coronifer (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Rebecchi, Lorena; V., Rossi; Altiero, Tiziana; Bertolani, Roberto; P., Menozzi

Allozymes were assessed by starch gel electrophoresis in 3 populations of a eutar-digrade, Richtersius coronifer, with different reproductive modes. One population from Italy (with 2 sub-populations) was amphimictic and 2 populations (1 from Italy and 1 from Sweden) were parthenogenetic. All populations, irrespective of their reproductive mode, were diploid with the same chromosome number (2n=12) and had bivalents in the oocytes. Of the 14 loci analyzed, only 3 were polymorphic. The amphimictic population had a higher degree of genetic variability (mean heterozygosity >0.25) than the parthenogenetic populations (mean heterozygosity of the 2 populations <0.01). In all female populations, allele frequencies at all 3 loci deviated from Hardy-Weinberg equilibria due to heterozygote deficiency. These results support a hypothesis of automictic parthenogenesis in R. coronifer.

2002 - Banding techniques on tardigrade chromosomes: the karyotype of Macrobiotus richtersi (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Rebecchi, Lorena; Altiero, Tiziana; Bertolani, Roberto

This work represents the first attempt to define tardigrade chromosomes using banding techniques. Macrobiotus richtersi, a eutardigrade morphospecies with amphimictic diploid and thelytokous triploid cytotypes, was used as a model. Prime consideration was given to oocyte chromosomes because they are larger than those of spermatocytes and of mitotic chromosomes. With Giemsa staining, the chromatids of the 6 bivalents of the diploid cytotypes and those of the 17 18 univalents of the triploid cytotypes were very similar to each other and appeared rod- or flame-shaped. In the amphimictic strain, a chiasma was generally present in each bivalent at diplotene, whereas there were no chiasmata in the oocyte prophase chromosomes of the triploid strain. Both in diploid and triploid cytotypes, C-banding and fluorescence showed a heterochromatic centromeric band on the telomere of each chromosome oriented towards the spindle pole, indicating that all of them were acrocentric. Silver staining showed the presence of a NOR in only a pair of chromosomes, close to the centromeric C-banded site. NOR was particularly evident in the oocyte prophases. Other silver positive regions, corresponding to the kinetochore, were located on all other chromosomes on the telomeres towards the spindle pole.

2001 - Rearing tardigrades: Results and problems [Articolo su rivista]
Altiero, Tiziana; Rebecchi, Lorena

We report our first results of attempts to rear four species of eutardigrades inhabiting different substrates, feeding on different kinds of food and characterized by different sexual conditions and modes of reproduction. Attempts were carried out to follow individual terrestrial carnivorous (Macrobiotus richtersi, M. joannae) and limnic herbivorous (Diphascon cf. scoticum; Isohypsibius monoicus) species. Carnivorous leaf litter-dwelling species were reared in small dishes containing agar as substrate and bacteriophagous nematodes as food. Five generations were obtained with the triploid. thelytokous strain of M. richtersi, whereas three generations were obtained with the hermaphrodite species M. joannae, Diphascon cf. scoticum and I. monoicus were reared in small dishes containing algae as food and substrate. Several generations were obtained for both species. Males were never found in D. cf. scoticum and I. monoicus was hermaphroditic. Specimens isolated from hatchings were maintained and reproduced in both species, demonstrating parthenogenesis in the first one and self-fertilization in the latter. Consideration of the problems and on the future applications of tardigrade rearing are discussed.

1998 - Chromosome c-banding and Ag-NOR pattern in tardigrades [Abstract in Atti di Convegno]
Rebecchi, Lorena; Altiero, Tiziana; Bertolani, Roberto

The first data on tardigrade chromosomes were derived from histological sections (Henneke 1911; von Wenck 1914). Specific studies were performed in the early seventies on animals stained in toto with acetic lactic orcein and squashed (Figs. 3-4; Bertolani 1971, 1975, 1982). Besides the definition of the chromosome number of several species (often n = 5 or n = 6), the main results were the identification of the polyploidy (triploidy and tetraploidy) and the definition of the cytology in the oocyte maturation of the parthenogenetic animals. Chromosomes always appeared small, without an evident centromere and similar to each other in the same plate and among the species. Oocyte chromosomes were clearly larger than those of the spermatocytes and of the mitotic divisions. More recently, Giemsa staining was applied to the eutardigrade Xerobiotus pseudohufelandi, in which diploid, triploid and tetraploid cytotypes were identified (Rebecchi, 1991). Triploidy and tetraploidy in tardigrades had been confirmed on the basis of the DNA content (Bertolani et al. 1987, 1994). Giemsa staining provides good details of the chromosome shape and confirms that M. richtersi is characterized by a chromosome set made up of very similar elements. Sex chromosomes are not recognizable.The kind of chromosome arrangement along the spindle fibers and the presence of a heterochromatic region on a telomere, evidenced by C-banding, allow us to conclude that all chromosomes of M. richtersi are acrocentric.There is only one NOR, localized on one extremity of one chromosome pair. It is evident in the oocyte prophases. In the oocyte metaphases the NOR could correspond to the most intense terminal dots that often characterize one of the six bivalents. As in other animals, the NOR coincides or is just adjacent the C-band site. The silver-positive regions located on one of the telomeres of all the other chromosomes of M. richtersi should correspond to the kynetochore, whereas the fainter regions located on the other telomere of all five bivalents resemble the “telochore” evidenced in the grasshoppers (Suja and Rufas 1994).