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Dipartimento di Scienze della Vita sede ex-Biologia

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2024 - The xerophilic genera Xerobiotus and Pseudohexapodibius (Macrobiotidae; Tardigrada): biodiversity, biogeography and phylogeny [Articolo su rivista]
Vincenzi, Joel; Cesari, Michele; Kaczmarek, ??ukasz; Roszkowska, Milena; Mioduchowska, Monika; Rebecchi, Lorena; Kiosya, Yevgen; Guidetti, Roberto

The genera Xerobiotus and Pseudohexapodibius are xerophilic and characterized by reduced appendages and claws as adaptations to move between small interstices. To increase the knowledge of their biodiversity, biogeography and phylogeny, several specimens from European countries and Australia were analysed using an integrative approach, i.e. morphological, karyological and molecular studies (18S, 28S, cox1, cytb and ITS2). The phylogenetic position of Xerobiotus with respect to the three evolutionary lineages of Macrobiotus remains unchanged with respect to what was previously identified in the phylogeny of Macrobiotidae. The following new Xerobiotus species are described: Xerobiotus litus sp. nov., Xerobiotus arenosum sp. nov. and Xerobiotus reductus sp. nov. It is proposed that Macrobiotus naginae should be transferred to Xerobiotus (Xerobiotus naginae comb. nov.). Pseudohexapodibius degenerans clusters within Xerobiotus and shares morphological and genetic characters with this genus. Some discrepancies between genetic and phenotypic data are found among the analysed species. Even between the results obtained with analysed genes disagreements are found, with cox1 and cytb generally agreeing with phenotypic results more than ITS2. Genotypic and phenotypic data are useful tools for species identification, but they have to be evaluated critically to obtain reliable results.

2023 - Ecology explains anhydrobiotic performance across tardigrades, but the shared evolutionary history matters more [Articolo su rivista]
Vecchi, M.; Stec, D.; Rebecchi, L.; Michalczyk, Ł.; Calhim, S.

Desiccation stress is lethal to most animals. However, some microinvertebrate groups have evolved coping strategies, such as the ability to undergo anhydrobiosis (i.e. survival despite the loss of almost all body water). Tardigrades are one such group, where the molecular mechanisms of anhydrobiosis have been more thoroughly studied. Despite the ecological, evolutionary and biotechnological importance of anhydrobiosis, little is known about its inter- and intra-specific variability nor its relationship with natural habitat conditions or phylogenetic history.We developed a new index-anhydrobiotic recovery index (ARI)-to evaluate the anhydrobiotic performance of tardigrade populations from the family Macrobiotidae. Moreover, we compared the explanatory role of habitat humidity and phylogenetic history on this trait using a variance partitioning approach.We found that ARI is correlated with both microhabitat humidity and yearly rainfall, but it is mostly driven by phylogenetic niche conservatism (i.e. a high portion of ARI variation is explained by phylogeny alone). Finally, we showed that anhydrobiotic performance is highly variable, even between closely related species, and that their response to local ecological conditions is tightly linked to their phylogenetic history.This study not only presents key insights into an emerging model system, but also provides a new methodological approach for wider scale studies of the ecological and evolutionary implications of anhydrobiosis.

2023 - Effects of synthetic acid rain and organic and inorganic acids on survival and CaCO3 piercing stylets in tardigrades [Articolo su rivista]
Massa, Edoardo; Rebecchi, Lorena; Guidetti, Roberto

Long-term environment acidifications due to decrease pH of the rainwaters affect both soils and water bodies. The organisms most likely to be affected by acid rain are the ones that possess vital organs made of calcium carbonate; among them are tardigrades, presenting aragonite piercing stylets in feeding apparatuses. A positive relationship between acidic rainfall and loss of tardigrades diversity has been already shown, but there is lack of knowledge of its lethal and sublethal effects. This study quantifies the effects of the acute exposure of three eutardigrade, Acutuncus antarcticus, Hypsibius exemplaris, and Macrobiotus cf. hufelandi, to synthetic acid rains and to organic and inorganic acids (hydrochloric, acetic, sulfuric, and nitric acids) naturally occurring in the environment. The cumulative proportion of dead animals in respect of exposition time was fitted to cumulative Weibull Distribution using a Bayesian framework. At the end of the experiments, animals were observed to investigate damages to their piercing stylets. Besides, stylets were finely morphologically described with Scanning Electron Microscopy. This study shows that acid rains and the other tested acids negatively affect tardigrades accordingly with pH, time of exposure, and tardigrade species. Freshwater species show a better resistance to acidity than the moss dwelling species, which can better acclimate over the time to low pH. The stylets resulted unaltered in almost all of the alive specimens. The results suggest that the tested tardigrades taxa have the ability to buffer the environmental proton change and the negative effect on their populations could be counteracted.

2023 - Increasing temperature-driven changes in life history traits and gene expression of an Antarctic tardigrade species [Articolo su rivista]
Giovannini, I.; Manfrin, C.; Greco, S.; Vincenzi, J.; Altiero, T.; Guidetti, R.; Giulianini, P.; Rebecchi, L.

The Antarctic region has been experiencing some of the planet's strongest climatic changes, including an expected increase of the land temperature. The potential effects of this warming trend will lead ecosystems to a risk of losing biodiversity. Antarctic mosses and lichens host different microbial groups, micro-arthropods and meiofaunal organisms (e.g., tardigrades, rotifers). The eutardigrade Acutuncus antarcticus is considered a model animal to study the effect of increasing temperature due to global warming on Antarctic terrestrial communities. In this study, life history traits and fitness of this species are analyzed by rearing specimens at two different and increasing temperatures (5 & DEG;C vs. 15 & DEG;C). Moreover, the first transcriptome analysis on A. antarcticus is performed, exposing adult animals to a gradual increase of temperature (5 & DEG;C, 10 & DEG;C, 15 & DEG;C, and 20 & DEG;C) to find differentially expressed genes under short- (1 day) and long-term (15 days) heat stress. Acutuncus antarcticus specimens reared at 5 & DEG;C live longer (maximum life span: 686 days), reach sexual maturity later, lay more eggs (which hatch in longer time and in lower percentage) compared with animals reared at 15 & DEG;C. The fitness decreases in animals belonging to the second generation at both rearing temperatures. The short-term heat exposure leads to significant changes at transcriptomic level, with 67 differentially expressed genes. Of these, 23 upregulated genes suggest alterations of mitochondrial activity and oxido-reductive processes, and two intrinsically disordered protein genes confirm their role to cope with heat stress. The long-term exposure induces alterations limited to 14 genes, and only one annotated gene is upregulated in response to both heat stresses. The decline in transcriptomic response after a long-term exposure indicates that the changes observed in the short-term are likely due to an acclimation response. Therefore, A. antarcticus could be able to cope with increasing temperature over time, including the future conditions imposed by global climate change.

2023 - The Macrobiotus persimilis-polonicus complex (Eutardigrada, Macrobiotidae), another example of problematic species identification, with the description of four new species. [Articolo su rivista]
Bertolani, Roberto; Cesari, Michele; Giovannini, Ilaria; Rebecchi, Lorena; Guidetti, Roberto; Kaczmarek, Łukasz; Pilato, Giovanni

In the phylum Tardigrada, as in other meiofauna taxa, the small size of the animals and the paucity of morphological characters useful for taxonomy present a challenge for systematic studies. For this reason, an integrated approach is increasingly desirable, including at least morphology, morphometry and molecular investigations, and possibly reproductive biology, ecology, physiology and other approaches. In this light, we analysed different populations of Macrobiotus (Eutardigrada, Macrobiotidae) from Italy, France and Poland with persimilis type of eggs and with very similar animals. We compared the morphology and morphometry of the type materials of Macrobiotus persimilis and Macrobiotus polonicus with those of newly collected populations, which were also analysed from a molecular (cox1 mtDNA and 18S rDNA) and reproductive perspective. Our data confirmed the presence of a persimilis-polonicus complex, recognizable by very similar characters of the animals and the eggs. It comprises M. persimilis, M. polonicus, four new morphologically distinguishable species described in this paper (Macrobiotus dolosus sp. nov., Macrobiotus siderophilus sp. nov., Macrobiotus fontourai sp. nov., Macrobiotus muralis sp. nov.) and other species very similar from a morphological point of view. Molecular data, retrieved for the first three new species, reveal that they have very high genetic distances for cox1 (16.0–17.7%). Furthermore, we have been able to verify that more than one species of this complex can cohabit in the same moss. A wider persimilis group, comprising the persimilis-polonicus complex, could also be identified only on morphological basis.

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 - Microhabitats, macro-differences: a survey of temperature records in Victoria Land terrestrial and freshwater environments [Articolo su rivista]
Cucini, Claudio; Nardi, Francesco; Magnoni, Letizia; Rebecchi, Lorena; Guidetti, Roberto; Convey, Peter; Carapelli, Antonio

The temperature experienced by micro-invertebrates in extreme environments (such as those of Antarctica) is a pivotal parameter regarding these animals' ecology and physiology. However, at present, detailed knowledge of microhabitat physical conditions in Antarctica is limited, as well as being biased towards sub-Antarctic and Maritime Antarctic regions. To better understand the temperature conditions experienced in the microhabitats of Continental Antarctica by the native microfauna, we recorded temperatures year round in ponds and soils in an area of the Victoria Land coast and compared these measurements with air temperature data from the closest automatic weather station. We identified an important difference in temperature dynamics between the air, soil and pond datasets. Ponds were the warmest sites overall, differing by up to 7.5°C in comparison with the air temperature due to their greater thermal capacity, which also drove their patterns of freeze-thaw cycles and mean daily thermal excursion.

2022 - Molecular Phylogenetics, Speciation, and Long Distance Dispersal in Tardigrade Evolution: A case study of the genusMilnesium [Articolo su rivista]
Guil, N; Guidetti, R; Cesari, M; Marchioro, T; Rebecchi, L; Machordom, A

: Microorganisms (sensu lato, i.e., including micrometazoans) are thought to have cosmopolitan geographic distributions due to their theoretically unlimited dispersal capabilities, a consequence of their tiny size, population dynamics, and resistant forms. However, several molecular studies of microorganisms have identified biogeographic patterns indicating cryptic speciation and/or weak species definitions. Using a multi-locus approach with the genus Milnesium (Tardigrada), we aimed to determine the genetic structure of populations worldwide and the effects of long distance dispersal (LDD) on genetic connectivity and relationships across the six continents. Our results on this micrometazoan's genetic structure and LDD at global and micro-local scales indicate contrasting patterns not easily explained by a unique or simple phenomenon. Overall, we report three key findings: (i) confirmation of long distance dispersal for tardigrades, (ii) populations with globally-shared or endemic micro-local haplotypes, and (iii) a supported genetic structure instead of the homogeneous genetic distribution hypothesized for microorganisms with LDD capabilities. Moreover, incongruences between our morphological and molecular results suggest that species delimitation within the genus Milnesium could be problematic due to homoplasy. Duality found for Milnesium populations at the global scale, namely, a molecular phylogenetic structure mixed with widely distributed haplotypes (but without any apparent biogeographic structure), is similar to patterns observed for some unicellular, prokaryotic and eukaryotic, microorganisms. Factors influencing these patterns are discussed within an evolutionary framework.

2022 - Morphology and taxonomy of the genus Ramazzottius (Eutardigrada; Ramazzottiidae) with the integrative description of Ramazzottius kretschmanni sp. nov [Articolo su rivista]
Guidetti, R.; Cesari, M.; Giovannini, I.; Ebel, C.; Forschler, M. I.; Rebecchi, L.; Schill, R. O.

The species of the genus Ramazzottius (Ramazzottiidae, Eutardigrada) are among the most common and widespread tardigrade species in the world. Most of the 28 Ramazzottius species have been described only with morphological characters which were most of the time represented only with drawings. The discovery of a new species of this genus in the Black Forest (Germany) provided the opportunity to compare this species with the type specimens of ten Ramazzottius species, to propose the status of species dubia for Ramazzottius edmondabouti, and through new photographs to elucidate the anatomy of animals and eggs (in particular of the head sensory regions, eye spots, buccal tube, ornamentations of the dorsal posterior cuticle, and morphology of egg processes). These thorough observations led to a better understanding of the diversity and evolution, not only of this cosmopolitan genus, but also of other eutardigrade genera. The new species Ramazzottius kretschmanni is described with an integrative approach integrating morphological (light and electron microscopy observations and morphometric data) and molecular (cox1 and ITS2 genes) data. The PTP and ASAP analyses confirmed the validity of the new species from a molecular point of view. The new species is morphologically similar to Ramazzottius oberhaeuseri, but is distinguishable by the smooth cuticle, the presence of a “cheek-like” area on the head, and the size of egg processes as well as different sequences of the molecular markers.

2022 - Phylogeny of the asexual lineage Murrayidae (Macrobiotoidea, Eutardigrada) with the description of Paramurrayon gen. nov. and Paramurrayon meieri sp. nov [Articolo su rivista]
Guidetti, Roberto; Giovannini, Ilaria; Del Papa, Valeria; Ekrem, Torbjørn; Nelson, Diane R.; Rebecchi, Lorena; Cesari, Michele

The peculiar family Murrayidae, comprising the genera Murrayon, Dactylobiotus and Macroversum, contains relatively rare species living in hydrophilic and freshwater habitats on all continents, and contains two of the six exclusively freshwater tardigrade genera. This family probably represents an example of the evolution and persistence of an asexual lineage that differentiated into several taxa without sexual reproduction. Analyses of nuclear and mitochondrial genes (18S, 28S, ITS2 and cox1), and the increase of five taxa to the phylogenetic analyses of Murrayidae led us to infer that Murrayon is polyphyletic, being composed of two ‘species groups’ that also find morphological supports: the ‘dianeae group’ characterised by peculiar egg processes (rod-shaped and covered with a cuticular layer), animals with large, evident epicuticular pillars and small claws; and the ‘pullari group’ characterised by conical egg processes, animals with very small epicuticular pillars, and proportionally larger and longer claws. This latter group is a sister group to Dactylobiotus. Murrayon hastatus is the only species within the genus that has an uncertain position with eggs of the ‘dianeae group’ and animals of the ‘pullari group’. We propose the erection of Paramurrayon gen. nov. (for the ‘dianeae group’ of species), the emendation of Murrayon, and new taxonomic keys for both genera. Possible scenarios of the evolution of taxa within Murrayidae are hypothesised based on synapomorphic characters. Paramurrayon meieri sp. nov. from Norway is described with an integrative approach. Photographs of type material of Murrayon stellatus, Murrayon nocentiniae, Murrayon ovoglabellus and Macroversum mirum are shown for the first time, together with descriptions of new characters. Murrayon hibernicus is considered as nomen dubium and Murrayon hyperoncus is transferred to Macrobiotus pending further analyses.

2022 - Production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in the tardigrade Paramacrobiotus spatialis [Articolo su rivista]
Giovannini, Ilaria; Boothby, Thomas C.; Cesari, Michele; Goldstein, Bob; Guidetti, Roberto; Rebecchi, Lorena

Water unavailability is an abiotic stress causing unfavourable conditions for life. Nevertheless, some animals evolved anhydrobiosis, a strategy allowing for the reversible organism dehydration and suspension of metabolism as a direct response to habitat desiccation. Anhydrobiotic animals undergo biochemical changes synthesizing bioprotectants to help combat desiccation stresses. One stress is the generation of reactive oxygen species (ROS). In this study, the eutardigrade Paramacrobiotus spatialis was used to investigate the occurrence of ROS associated with the desiccation process. We observed that the production of ROS signifcantly increases as a function of time spent in anhydrobiosis and represents a direct demonstration of oxidative stress in tardigrades. The degree of involvement of bioprotectants, including those combating ROS, in the P. spatialis was evaluated by perturbing their gene functions using RNA interference and assessing the successful recovery of animals after desiccation/rehydration. Targeting the glutathione peroxidase gene compromised survival during drying and rehydration, providing evidence for the role of the gene in desiccation tolerance. Targeting genes encoding glutathione reductase and catalase indicated that these molecules play roles during rehydration. Our study also confrms the involvement of aquaporins 3 and 10 during rehydration. Therefore, desiccation tolerance depends on the synergistic action of many diferent molecules working together.

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 - Comparative phylogeography reveals consistently shallow genetic diversity in a mitochondrial marker in Antarctic bdelloid rotifers [Articolo su rivista]
Cakil, Z. V.; Garlasche, G.; Iakovenko, N.; Di Cesare, A.; Eckert, E. M.; Guidetti, R.; Hamdan, L.; Janko, K.; Lukashanets, D.; Rebecchi, L.; Schiaparelli, S.; Sforzi, T.; Kasparova, E. S.; Velasco-Castrillon, A.; Walsh, E. J.; Fontaneto, D.

Aim: The long history of isolation of the Antarctic continent, coupled with the harsh ecological conditions of freezing temperatures, could affect the patterns of genetic diversity in the organisms living there. We aim (a) to test whether such pattern can be seen in a mitochondrial marker of bdelloid rotifers, a group of microscopic aquatic and limno-terrestrial animals and (b) to speculate on the potential mechanisms driving the pattern. Location: Focus on Antarctica. Taxon: Rotifera Bdelloidea. Methods: We analysed different metrics of genetic diversity, also spatially explicit ones, including number of haplotypes, accumulation curves, genetic distances, time to the most recent common ancestor, number of independently evolving units from DNA taxonomy, strength of the correlation between geographical and genetic distances, population genetics neutrality and differentiation indices, potential historical processes, obtained from an extensive sample of cytochrome oxidase subunit I (COI) sequences obtained from bdelloid rotifers. We included 2242 individuals from 23 species in a comparison between Antarctic and non-Antarctic taxa, correcting for sample size directly in the analyses and then by confirming the results also using only a restricted dataset of nine well-sampled species. Results: Antarctic species had consistently lower genetic diversity and potential younger relative age than non-Antarctic species, even if they were similar in sample size, geographical extent, neutrality and differentiation indices, and correlation between genetic and geographical distances. Main conclusions: The extensive survey of genetic diversity in one mitochondrial marker in Antarctic bdelloids supports previous suggestions from other organisms that the origin and maintenance of terrestrial Antarctic fauna are different from those of other continents. Such differences could be speculated to be due, in the case of bdelloid rotifers, to the more recent origin of the species living there in comparison to non-Antarctic species.

2021 - Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. (Eutardigrada, Macrobiotidae, hufelandi group) [Articolo su rivista]
Ben Marnissi, Jamila; Cesari, Michele; Rebecchi, Lorena; Bertolani, Roberto

2021 - Tardigrades of Kristianstads Vattenrike Biosphere Reserve with description of four new species from Sweden [Articolo su rivista]
Massa, Edoardo; Guidetti, Roberto; Cesari, Michele; Rebecchi, Lorena; Jönsson, K. Ingemar

Kristianstads Vattenrike Biosphere Reserve [KVBR] is a UNESCO designated area of Sweden possessing high biological value. Although several studies on tardigrades inhabiting Sweden have been performed, the KVBR area has been neglected. The current study investigates the tardigrade fauna of five areas of the biosphere reserve and includes 34 samples of different substrates analysed quantitatively and qualitatively. In total, 33 species of tardigrades were found in the samples, including 22 new records for the Skåne region, 15 new records for Sweden, and four species new to science. Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov. were described with an integrative approach (when possible) using morphological characters (light, electron scanning, and confocal laser scanning microscopies) and molecular markers (ITS2, 18S, 28S, cox1). A new protocol to increase morphological data was developed recovering mounted specimens within old slides for SEM analysis. Emended diagnoses for the genus Itaquascon and the transfer of Platicrista itaquasconoide to the genus Meplitumen are proposed. This study enriches the knowledge of the tardigrade biodiversity both within the KVBR and in Sweden and contributes to the rapidly increasing number of tardigrade species reported worldwide. The 33 species identified in the KVBR area represents 28% of all water bear species found in Sweden so far. The restricted study areas and limited number of samples collected suggests that the KVBR is very rich of tardigrades.

2021 - The toughest animals of the Earth versus global warming: Effects of long-term experimental warming on tardigrade community structure of a temperate deciduous forest [Articolo su rivista]
Vecchi, M.; Kossi Adakpo, L.; Dunn, R. R.; Nichols, L. M.; Penick, C. A.; Sanders, N. J.; Rebecchi, L.; Guidetti, R.

Understanding how different taxa respond to global warming is essential for predicting future changes and elaborating strategies to buffer them. Tardigrades are well known for their ability to survive environmental stressors, such as drying and freezing, by undergoing cryptobiosis and rapidly recovering their metabolic function after stressors cease. Determining the extent to which animals that undergo cryptobiosis are affected by environmental warming will help to understand the real magnitude climate change will have on these organisms. Here, we report on the responses of tardigrades within a five-year-long, field-based artificial warming experiment, which consisted of 12 open-top chambers heated to simulate the projected effects of global warming (ranging from 0 to 5.5°C above ambient temperature) in a temperate deciduous forest of North Carolina (USA). To elucidate the effects of warming on the tardigrade community inhabiting the soil litter, three community diversity indices (abundance, species richness, and Shannon diversity) and the abundance of the three most abundant species (Diphascon pingue, Adropion scoticum, and Mesobiotus sp.) were determined. Their relationships with air temperature, soil moisture, and the interaction between air temperature and soil moisture were tested using Bayesian generalized linear mixed models. Despite observed negative effects of warming on other ground invertebrates in previous studies at this site, long-term warming did not affect the abundance, richness, or diversity of tardigrades in this experiment. These results are in line with previous experimental studies, indicating that tardigrades may not be directly affected by ongoing global warming, possibly due to their thermotolerance and cryptobiotic abilities to avoid negative effects of stressful temperatures, and the buffering effect on temperature of the soil litter substrate.

2021 - When DNA sequence data and morphological results fit together: Phylogenetic position of Crenubiotus within Macrobiotoidea (Eutardigrada) with description of Crenubiotus ruhesteini sp. nov [Articolo su rivista]
Guidetti, R.; Schill, R. O.; Giovannini, I.; Massa, E.; Goldoni, S. E.; Ebel, C.; Forschler, M. I.; Rebecchi, L.; Cesari, M.

The integration of morphological data and data from molecular genetic markers is important for examining the taxonomy of meiofaunal animals, especially for eutardigrades, which have a reduced number of morphological characters. This integrative approach has been used more frequently, but several tardigrade taxa lack molecular confirmation. Here, we describe Crenubiotus ruhesteini sp. nov. from the Black Forest (Germany) integratively, with light and electron microscopy and with sequences of four molecular markers (18S, 28S, ITS2, cox1 genes). Molecular genetic markers were also used to confirm the recently described Crenubiotus genus and to establish its phylogenetic position within the Macrobiotoidea (Eutardigrada). The erection of Crenubiotus and its place in the family Richtersiidae are confirmed. Richtersiidae is redescribed as Richtersiusidae fam. nov. because its former name was a junior homonym of a nematode family.

2020 - A data set on the distribution of Rotifera in Antarctica [Articolo su rivista]
Garlasché, Giuseppe; Karimullah, Karimullah; Iakovenko, Nataliia; Velasco-Castrillón, Alejandro; Janko, Karel; Guidetti, Roberto; Rebecchi, Lorena; Cecchetto, Matteo; Schiaparelli, Stefano; Jersabek, Christian D.; De Smet, Willem H.; Fontaneto, Diego

We present a data set on Antarctic biodiversity for the phylum Rotifera, making it publicly available through the Antarctic Biodiversity Information facility. We provide taxonomic information, geographic distribution, location, and habitat for each record. The data set gathers all the published literature about rotifers found and identified across the Continental, Maritime, and Subantarctic biogeographic regions of Antarctica. A total of 1455 records of rotifers in Antarctica found from 1907 to 2018 is reported, with information on taxonomic hierarchies, updated nomenclature, geographic information, geographic coordinates, and type of habitat. The aim is to provide a georeferenced data set on Antarctic rotifers as a baseline for further studies, to improve our knowledge on what has been considered one of the most diverse and successful groups of animals living in Antarctica.

2020 - An integrated study of the biodiversity within the Pseudechiniscus suillus–facettalis group (Heterotardigrada: Echiniscidae) [Articolo su rivista]
Cesari, Michele; Montanari, Martina; Kristensen, Reinhardt M; Bertolani, Roberto; Guidetti, Roberto; Rebecchi, Lorena

Pseudechiniscus is the second most species-rich genus in Heterotardigrada and in the family Echiniscidae. However, previous studies have pointed out polyphyly and heterogeneity in this taxon. The recent erection of the genus Acanthechiniscus was another step in making Pseudechiniscus monophyletic, but species identification is still problematic. The present investigation aims at clarifying biodiversity and taxonomy of Pseudechiniscus taxa, with a special focus on species pertaining to the so-called ‘suillus–facettalis group’, by using an integrated approach of morphological and molecular investigations. The analysis of sequences from specimens sampled in Europe and Asia confirms the monophyly of the genus Pseudechiniscus. Inside the genus, two main evolutionary lineages are recognizable: the P. novaezeelandiae lineage and the P. suillus–facettalis group lineage. Inside the P. suillus–facettalis group, COI molecular data points out a very high variability between sampled localities, but in some cases also among specimens sampled in the same locality (up to 33.3% p-distance). The integrated approach to the study of Pseudechiniscus allows confirmation of its monophyly and highlights the relationships in the taxon, pointing to its global distribution.

2020 - Extreme-tolerance mechanisms in meiofaunal organisms: a case study with tardigrades, rotifers and nematodes [Articolo su rivista]
Rebecchi, L.; Boschetti, C.; Nelson, D. R.

To persist in extreme environments, some meiofaunal taxa have adopted outstanding resistance strategies. Recent years have seen increased enthusiasm for understanding extreme-resistance mechanisms evolved by tardigrades, nematodes and rotifers, such as the capability to tolerate complete desiccation and freezing by entering a state of reversible suspension of metabolism called anhydrobiosis and cryobiosis, respectively. In contrast, the less common phenomenon of diapause, which includes encystment and cyclomorphosis, is defined by a suspension of growth and development with a reduction in metabolic activity induced by stressful environmental conditions. Because of their unique resistance, tardigrades and rotifers have been proposed as model organisms in the fields of exobiology and space research. They are also increasingly considered in medical research with the hope that their resistance mechanisms could be used to improve the tolerance of human cells to extreme stress. This review will analyse the dormancy strategies in tardigrades, rotifers and nematodes with emphasis on mechanisms of extreme stress tolerance to identify convergent and unique strategies occurring in these distinct groups. We also examine the ecological and evolutionary consequences of extreme tolerance by summarizing recent advances in this field.

2020 - Further insights in the Tardigrada microbiome: phylogenetic position and prevalence of infection of four new Alphaproteobacteria putative endosymbionts [Articolo su rivista]
Rebecchi, Lorena; Cesari, Michele; Newton, Irene L G; Ferrari, Agnese; Vecchi, Matteo; Guidetti, Roberto

Data from a previous study showed that microbiomes of six tardigrade species are species-specific and distinct from associated environmental microbes. We here performed a more in-depth analyses of those data, to identify and characterize new potential symbionts. The most abundant bacterial operational taxonomic units (OTUs) found in tardigrades are classified, and their prevalence in other environments is assessed using public databases. A subset of OTUs was selected for molecular phylogenetic analyses based on their affiliation with host-associated bacterial families in tardigrades. Almost 22.6% of the most abundant OTUs found do not match any sequence at 99% identity in the IMNGS database. These novel OTUs include four putative tardigrade endosymbionts from Alphaproteobacteria (Anaplasmataceae and Candidatus Tenuibacteraceae), which are characterized by 16S rRNA gene analysis and investigated for their infection rates in: Echiniscus trisetosus, Richtersisus coronifer and Macrobiotus macrocalix. These putative endosymbionts have an infection prevalence between 9.1% and 40.0%, and are, therefore, likely secondary symbionts, not essential for tardigrade survival and reproduction. Using fluorescence in situ hybridization (FISH), we detected bacteria on the cuticle and within the ovary of E. trisetosus, suggesting possible vertical transmission. This study highlights the great contribution in biodiversity discovery that neglected phyla can provide in microbiome and symbiosis studies.

2020 - Phylum Tardigrada [Capitolo/Saggio]
Nelson, Diane R.; Guidetti, Roberto; Rebecchi, Lorena; Kaczmarek, Łukasz; Mcinnes, Sandra

2020 - The species identification problem in mirids (Hemiptera: Heteroptera) highlighted by DNA barcoding and species delimitation studies [Articolo su rivista]
Piemontese, L.; Giovannini, I.; Guidetti, R.; Pellegri, G.; Dioli, P.; Maistrello, L.; Rebecchi, L.; Cesari, M.

Due to the difficulties associated with detecting and correctly identifying mirids, developing an accurate species identification approach is crucial, especially for potential harmful species. Accurate identification is often hampered by inadequate morphological key characters, invalid and/or outdated systematics, and biases in the molecular data available in public databases. This study aimed to verify whether molecular characterization (i.e. DNA barcoding) is able to identify mirid species of economic relevance and if species delimitation approaches are reliable tools for species discrimination. Cytochrome c oxydase 1 (cox1) data from public genetic databases were compared with new data obtained from mirids sampled in different Italian localities, including an old specimen from private collection, showing contrasting results. Based on the DNA barcoding approach, for the genus Orthops, all sequences were unambiguously assigned to the same species, while in Adelphocoris, Lygus and Trigonotylus there were over-descriptions and/or misidentifications of species. On the other hand, in Polymerus and Deraeocoris there was an underestimation of the taxonomic diversity. The present study highlighted an important methodological problem: DNA barcoding can be a good tool for pest identification and discrimination, but the taxonomic unreliability of public DNA databases can make this method useless or even misleading.

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.

2019 - Increasing knowledge of Antarctic biodiversity: new endemic taxa of tardigrades (Eutardigrada; Ramazzottiidae) and their evolutionary relationships [Articolo su rivista]
Guidetti, R.; Massa, Edoardo; Bertolani, R.; Rebecchi, L.; Cesari, M.

The underestimation of biodiversity and scarce knowledge of the biogeographic distributions of terrestrial meiofaunal component limit our understanding of the origin of Antarctic fauna. One of its main components is tardigrades; nonetheless studies on tardigrade diversity are still few in the continental Antarctic area. In order to increase our knowledge of the underreported terrestrial meiofaunal communities within continental Antarctica, as well as to provide new information for biogeographic and evolutionary analysis of these communities, the tardigrade diversity of 11 samples collected along the coast of Victoria Land was considered. The application of an integrative approach in which morphological (Light - LM, scanning electron - SEM, and confocal laser scanning microscopy - CSLM) and molecular analysis (18S, 28S, cox1 genes) were combined allowed us to characterize the tardigrade fauna inhabiting mosses, lichens, and cyanobacterial mats of Victoria Land. These analyses allowed the detection of two tardigrade species new to science (Cryoconicus antiarktos sp. nov., Ramazzottius sabatiniae sp. nov.), and the emendation of known species ((Hebesuncus ryani Dastych and Harris, 1994, Ramazzottius nivalis Dastych, 2006, Cryoconicus ljudmilae comb. nov. (Biserov, 1997/98), and Cryoconicus cataphractus (Maucci, 1974)), together with the genus Cryoconicus. The Ramazzottius type of claws was redefined identifying three subtypes. Also, we investigated the phylogenetic position of some problematic/unresolved lineages all belonging to the family Ramazzottiidae. These data increased the knowledge of the biodiversity in Victoria Land, the number of endemic tardigrades in Antarctica, and provided evidence on the origin of Antarctic endemism. Lastly, new methods for integrative taxonomic studies on tardigrades were presented and discussed.

2019 - Phylum Tardigrada [Capitolo/Saggio]
Nelson, R. Diane; Guidetti, Roberto; Rebecchi, Lorena

The chapter shows anatomy, geographical distribution and taxonomical key of tardigrades from the palearctic region.

2018 - Cytology and Cytogenetics [Capitolo/Saggio]
Bertolani, Roberto; Rebecchi, Lorena

Several cytological aspects have been considered in tardigrades. Firstly, the cell constancy which is not a true eutely being several mitoses present even after hatching, even though some organs, such epidermis and nervous ganglia, have the same cell number in juveniles and adults. The total number of these cells is speciesspecific. Then the ultrastructure of cuticle, epidermis, feeding and digestive apparatus, excretory and osmoregulatory organs, muscles, nerve cells, sensory cells and storage cells has been considered. Instead, the ultrastructure of the germ cells has been considered in the chapter on reproduction. With regard to chromosome number and shape, it has been observed that generally there is little difference among the species (n ¼ 5 or n ¼ 6), but several cases of polyploid populations exist, often very similar to diploid populations from a morphological point of view. In most cases the polyploid populations do not have males and reproduce by apomixis. Studies on the genome size have confirmed the presence of polyploid populations, as well as the presence of nuclei with multiple amounts of DNA within the same specimen. The genome size of the tardigrades is always relatively small and does not seem related to phylogenetic lineages. Studies on tardigrade genomes have placed this phylum at the centre of discussions on the evolution of Metazoa and have considered the role of horizontal gene transfer in animal evolution with contrasting results.

2018 - Genetic diversity of the brown marmorated stink bug Halyomorpha halys in the invaded territories of Europe and its patterns of diffusion in Italy [Articolo su rivista]
Cesari, Michele; Maistrello, Lara; Piemontese, Lucia; Bonini, Raoul; Dioli, Paride; Lee, Wonhoon; Park, Chang-Gyu; Partsinevelos, Georgios K.; Rebecchi, Lorena; Guidetti, Roberto

Halyomorpha halys is an invasive stink bug pest originating from East Asia. In Europe, it was first detected in Switzerland in 2004. It is now present in thirteen countries, and seems to be spreading throughout the continent. In Italy, where it has been recorded since 2012, other than being an urban nuisance, it is causing severe damage in commercial fruit orchards. An integrated approach, using current and previous observational data in space and time and molecular information, was used to identify the genetic diversity of this pest in Europe, its invasion history, and the potential pathways of entry and diffusion. The analysis of 1175 bp of mitochondrial DNA cytochrome c oxidase I and II genes (cox1, cox2) led to the identification of twenty previously unknown haplotypes. The European distribution of H. halys is the result of multiple invasions that are still in progress, and, in some cases, it was possible to identify the specific Asian areas of origin. Moreover, secondary invasions could have occurred among European countries by a bridgehead effect. In Italy, the data were more clearly related to their temporal occurrence, allowing for a clearer reading of the patterns of invasion and dispersion. After having successfully established in localized areas, H. halys further expanded its range by an active dispersion process and/or by jump dispersal events due to passive transport. The multiple introductions from different areas of the native range together with the different patterns of diffusion of H. halys, may hamper the pest management strategies for its containment.

2018 - Molecular palaeontology illuminates the evolution of ecdysozoan vision [Articolo su rivista]
Fleming, James F; Kristensen, Reinhardt Møbjerg; Sørensen, Martin Vinther; Park, Tae-Yoon S; Arakawa, Kazuharu; Blaxter, Mark; Rebecchi, Lorena; Guidetti, Roberto; Williams, Tom A; Roberts, Nicholas W; Vinther, Jakob; Pisani, Davide

Colour vision is known to have arisen only twice-once in Vertebrata and once within the Ecdysozoa, in Arthropoda. However, the evolutionary history of ecdysozoan vision is unclear. At the molecular level, visual pigments, composed of a chromophore and a protein belonging to the opsin family, have different spectral sensitivities and these mediate colour vision. At the morphological level, ecdysozoan vision is conveyed by eyes of variable levels of complexity; from the simple ocelli observed in the velvet worms (phylum Onychophora) to the marvellously complex eyes of insects, spiders, and crustaceans. Here, we explore the evolution of ecdysozoan vision at both the molecular and morphological level; combining analysis of a large-scale opsin dataset that includes previously unknown ecdysozoan opsins with morphological analyses of key Cambrian fossils with preserved eye structures. We found that while several non-arthropod ecdysozoan lineages have multiple opsins, arthropod multi-opsin vision evolved through a series of gene duplications that were fixed in a period of 35-71 million years (Ma) along the stem arthropod lineage. Our integrative study of the fossil and molecular record of vision indicates that fossils with more complex eyes were likely to have possessed a larger complement of opsin genes.

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 - The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome Structure and Composition [Articolo su rivista]
Vecchi, Matteo; Newton, Irene L. G.; Cesari, Michele; Rebecchi, Lorena; Guidetti, Roberto

Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations.We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.

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.

2017 - Evolutionary scenarios for the origin of an Antarctic tardigrade species based on molecular clock analyses and biogeographic data [Articolo su rivista]
Guidetti, Roberto; Mcinnes, J. Sandra; Cesari, Michele; Rebecchi, Lorena; Rota Stabelli, Omar

The origin of the Antarctic continental extant fauna is a highly debated topic, complicated by the paucity of organisms for which we have clear biogeographic distributions and understanding of their evolutionary timescale. To shed new light on this topic, we coupled molecular clock analyses with biogeographic studies on the heterotardigrade genus Mopsechiniscus. This taxon includes species with endemic distributions in Antarctica and other regions of the southern hemisphere. Molecular dating using different models and calibration priors retrieved similar divergence time for the split between the Antarctic and South American Mopsechiniscus lineages (32–48 Mya) and the estimated age of the Drake Passage opening that led to the separation of Antarctica and South America. Our divergence estimates are congruent with other independent studies in dating Gondwanan geological events. Although different analyses retrieved similar results for the internal relationships within the Heterotardigrada, our results indicated that the molecular dating of tardigrades using genes coding for ribosomal RNA (18S and 28S rDNA) is a complex task, revealed by a very wide range of posterior density and a relative difficulty in discriminating between competing models. Overall, our study indicates that Mopsechiniscus is an ancient genus with a clear Gondwanan distribution, in which speciation was probably directed by a cooccurrence of vicariance and glacial events.

2017 - Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation [Articolo su rivista]
Boothby, Thomas C.; Tapia, Hugo; Brozena, Alexandra H.; Piszkiewicz, Samantha; Smith, Austin E; Giovannini, Ilaria; Rebecchi, Lorena; Pielak, Gary J.; Koshland, Dough; Goldstein, Bob

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

2016 - Comparative analysis of fatty acid profile in three eutardigrade species [Poster]
Giovannini, I; Mantovani, V; Galeotti, F; Chersoni, L; Guidetti, R; Volpi, N; Rebecchi, L.

Tardigrades colonize a wide range of habitats in which they can be predators, prey or primary consumers in food webs. Most species are herbivorous, feeding on cell fluid of algae and mosses, while others feed on bacteria, or prey on micrometazoans. Despite the wide range of food sources, details on food preference and on consequent lipid composition of tardigrade species are in practice unknown. Aiming to fill the gap of knowledge, we investigated the fatty acid composition of three eutardigrade species, since fatty acids are the main component of lipids and they play an important role in the function of cell membranes and in the physiological responses of organisms. The species, differing in colonized habitat and probably in diet, were: Acutuncus antarcticus (Hypsibiidae), a freshwater Antarctic species cultured using Chlorococcum sp. as food source, and the moss-dwelling species Macrobiotus macrocalix and Richtersius coronifer (Macrobiotidae). For each species, lipids were extracted from ten replicates of 150-250 animals with chloroform/methanol and the total extracts were used to obtain the fatty acid metylesters that were injected into a gas chromatograph. In all species, the same 21 fatty acids belonging to saturated, monounsaturated (MUFA) and polyunsaturated (PUFA) groups were identified. In A. antarcticus the most represented fatty acids were: palmitic (C16:0), stearic (C18:0), oleic (C18:1n-9), and myristic (C14:0) acids; saturated fatty acids (56.6%) were the most abundant with respect to MUFA (22.3%) and PUFA (21.1%). In M. macrocalix the most represented were: oleic (C18:1n-9), palmitic (C16:0), stearic (C18:0), and linoleic (C18:2n-6) acids; the saturated fatty acids (38.4%), MUFA (28.8%) and PUFA (32.8%) were uniformly distributed. In R. coronifer, alpha-linolenic (C18:3n-3), palmitic (C16:0), stearic (C18:0), and arachidonic (C20:4n-6) acids are the most represented; the percentage of PUFA (52.8%) was higher than that of MUFA (8.2%) and saturated fatty acids (38.9%). These data indicate clear differences in the fatty acid composition and amount among species. The fatty acid profiles reflect the food source and can be used as indicator to assess the feeding diet of tardigrades. Interestingly, species inhabiting the same substrate and eating the same food (moss cell content) use/transform the fatty acids in different way indicating different biochemical needs.

2016 - Genetic diversity and biogeography of the south polar water bear Acutuncus antarcticus (Eutardigrada : Hypsibiidae) – evidence that it is a truly pan-Antarctic species [Articolo su rivista]
Cesari, Michele; Mcinnes, J. Sandra; Bertolani, Roberto; Rebecchi, Lorena; Guidetti, Roberto

Antarctica is an ice-dominated continent and all its terrestrial and freshwater habitats are fragmented, which leads to genetic divergence and, eventually, speciation. Acutuncus antarcticus is the most common Antarctic tardigrade and its cryptobiotic capabilities, small size and parthenogenetic reproduction present a high potential for dispersal and colonisation. Morphological (light and electron microscopy, karyology) and molecular (18S rRNA and cytochrome c oxidase subunit I (COI) genes) analyses on seven populations of A. antarcticus elucidated the genetic diversity and distribution of this species. All analysed populations were morphologically indistinguishable and made up of diploid females. All specimens presented the same 18S rRNA sequence. In contrast, COI analysis showed higher variability, with most Victoria Land populations presenting up to five different haplotypes. Genetic distances between Victoria Land specimens and those found elsewhere in Antarctica were low, while distances between Dronning Maud Land and specimens from elsewhere were high. Our analyses show that A. antarcticus can still be considered a pan-Antarctic species, although the moderately high genetic diversity within Victoria Land indicates the potential for speciation events. Regions of Victoria Land are considered to have been possible refugia during the last glacial maximum and a current biodiversity hotspot, which the populations of A. antarcticus mirror with a higher diversity than in other regions of Antarctica.

2016 - Integrative systematic studies on tardigrades from Antarctica identify new genera and new species within Macrobiotoidea and Echiniscoidea [Articolo su rivista]
Vecchi, Matteo; Cesari, Michele; Bertolani, Roberto; Jonsson, K. Ingemar; Rebecchi, Lorena; Guidetti, Roberto

Tardigrades represent one of the most abundant groups of Antarctic metazoans in terms of abundance and diversity, thanks to their ability to withstand desiccation and freezing; however, their biodiversity is underestimated. Antarctic tardigrades from Dronning Maud Land and Victoria Land were analysed from a morphological point of view with light microscopy and scanning electron microscopy, and from a molecular point of view using two genes (18S, 28S) analysed in Bayesian inference and maximum-likelihood frameworks. In addition, indel-coding datasets were used for the first time to infer tardigrade phylogenies. We also compared Antarctic specimens with those from Italy and Greenland. A combined morphological and molecular analysis led to the identification of two new evolutionary lineages, for which we here erect the new genera Acanthechiniscus, gen. nov. (Echiniscidae, Echiniscoidea) and Mesobiotus, gen. nov. (Macrobiotidae, Macrobiotoidea). Moreover, two species new to science were discovered: Pseudechiniscus titianae, sp. nov. (Echiniscidae : Echiniscoidea) and Mesobiotus hilariae, sp. nov. (Macrobiotidae : Macrobiotoidea). This study highlights the high tardigrade diversity in Antarctica and the importance of an integrated approach in faunal and taxonomic studies.

2016 - Interspecific relationships of tardigrades with bacteria, fungi and protozoans, with a focus on the phylogenetic position of Pyxidium tardigradum (Ciliophora) [Articolo su rivista]
Vecchi, Matteo; Vicente, Filipe; Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele

Symbiosis can be defined as an interaction between individuals of different biological species. Despite the small number of studies solely devoted to symbiotic interactions between tardigrades and micro-organisms (such as bacteria, fungi and protozoans), numerous reports can be found in the literature, especially as notes in faunal and alpha-taxonomy studies. Here, we review the literature and compile a list of the interactions between tardigrades and micro-organisms, excluding those that constitute food for tardigrades. Furthermore, a genetic study on a tardigrade symbiophoront, the ciliate Pyxidium tardigradum van der Land, 1964 was performed. There are a few records of P. tardigradum on both European and Asian tardigrades, but no morphological discrepancies among populations have been observed. We present here the phylogenetic positioning for P. tardigradum inferred by using nuclear ribosomal markers (18S and 5.8S). The phylogenetic trees showed all P. tardigradum specimens grouped together, and belonging to the family Operculariidae, order Operculariida. Moreover, a study based on the genetic distances between a Portuguese and an Irish population of P. tardigradum was performed using internal transcribed spacers ITS1 and ITS2. The ITS1 and ITS2 sequences showed differences between populations, leading us to hypothesize the presence of cryptic species.

2016 - Introductory Text – Preface [Breve Introduzione]
Rebecchi, Lorena; Diane R., Nelson

The proceedings of the 13th International Symposium on Tardigrada contain 23 peer-reviewed papers, published as an issue of the Zoological Journal of the Linnean Society. In the current issue, the papers are diversified and engaging. They cover a wide range of themes such as taxonomy, phylogeny, biogeography, ecology, reproductive and developmental biology, biochemistry, adaptive strategies, genetics, genomics and metabolomics.

2016 - Morphological and molecular analyses on Richtersius (Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea [Articolo su rivista]
Guidetti, Roberto; Rebecchi, Lorena; Bertolani, Roberto; Jönsson, K. Ingemar; Kristensen, M. Reinhardt; Cesari, Michele

Important contributions have been made to the systematics of Eutardigrada in recent years, but these have also revealed that several taxa are polyphyletic and that cryptic species are present. To shed light on the taxonomy and systematic position of the genus Richtersius (Eutardigrada, Macrobiotoidea), six populations attributed to Richtersius coronifer were collected and analysed from morphological (light and scanning electron microscopy) and molecular (mitochondrial cytochrome oxidase subunit 1, 18S, 28S) points of view. In particular, a new morphometric index (claw common tract: length of the common tract of the claw/total claw length 9 100) and a new morphological character (stalk system) were introduced. Our integrative study was able to unveil the ‘cryptic’ species diversity within Richtersius, showing that the genus contains more than one evolutionary lineage. A morphological peculiarity in the animals of all lineages is the dimorphism in the morphology of the cuticle. Cuticular pores are present in the newborns and are lost with the first moult; this morphological change represents a novelty in the life cycle of eutardigrades. The phylogenetic analyses carried out on Richtersius populations and other Macrobiotoidea show that Richtersius is closely related to Macrobiotus islandicus, whereas Adorybiotus granulatus is more related to Richtersius and M. islandicus than to other members of the genus Macrobiotus (type genus of Macrobiotidae); therefore, the genus Macrobiotus and the family Macrobiotidae are not monophyletic. Based on these results, the new genus Diaforobiotus (for M. islandicus) and the new family Richtersiidae (composed of Richtersius, Diaforobiotus gen. nov., and Adorybiotus) are established.

2016 - New multivariate image analysis method for detection of differences in chemical and structural composition of chitin structures in tardigrade feeding apparatuses [Articolo su rivista]
Savic, Aleksandar G; Preus, Soren; Rebecchi, Lorena; Guidetti, Roberto

The feeding apparatus of the eutardigrade Paramacrobiotus richtersi was examined with confocal laser scanning microscopy using three different wavelengths, taking advantage of the autofluorescence of the chitin present in its structures. The main goal was to test new method based on multivariate image analysis for detection of differences in the chemical and structural composition of chitin structures and the relationships between chemical structure and physical properties of feeding structures. The collected images were analyzed using factor analysis under the self-developed MATLABbased software ImageA. It was possible to observe small differences in chitin composition using the variations (shifts) of emission spectra caused by differences in the microenvironment of chitin fluorophore. Images of feeding apparatuses within the body and discharged by the animal during molt were recorded applying three excitation/ emission (ex/em) sets and then analyzed using factor analysis with three methods for rotation of factor scores (without rotation, with orthogonal rotation, and oblique rotation). With this procedure, it was proven that chitin organization in the feeding apparatus of tardigrades is not homogeneous, but changes according to the mechanical and structural requirements of the structures. Besides the new knowledge about the tardigrade feeding apparatus, ImageA was also proven to be reliable and applicable tool for various problems in the analysis of multispectral images, as autofluorescence images are generally considered as difficult to analyze due to the small initial set of input data and high correlation among the images observed at different ex/em wavelengths

2016 - Phylum Tardigrada. 15 [Capitolo/Saggio]
Nelson, Diane; Guidetti, Roberto; Rebecchi, Lorena

The chapter reports the systematics, ecology, distribution and keys of freshwater tardigrades of nearctic biogeographical region.

2016 - Specie aliene invasive: il caso della cimice bruna marmorizzata Halyomorpha halys (Heteroptera, Pentatomidae) in Italia e nel territorio modenese [Articolo su rivista]
Piemontese, Lucia; Cesari, Michele; Ganzerli, Francesco; Maistrello, Lara; Dioli, Paride; Rebecchi, Lorena; Guidetti, Roberto

Invasive alien species: the case of the brown marmorated stink bug (Halyomorpha halys) in Italy. Organisms that intentionally or unintentionally are introduced by human activities in a region beyond their native range, can give rise to biological invasions of alien species that are a threat to biodiversity, agriculture, economy and public health. In this work, the biological and evolutionary aspects related to these events and the molecular methods used in the field of Invasion Biology studies are taken into account. In addition, a new invasive species in Europe and Italy is analysed: the brown marmorated stink bug (Halyomorpha halys), an agricultural and urban pest native to East Asia. Considering the economic losses in the province of Modena caused by this insect during 2015, the results on the origins, invasion pathways and dispersal patterns of the populations analysed can be useful in order to implement better pest control and/or prevention strategies.

2016 - The Compact Body Plan of Tardigrades Evolved by the Loss of a Large Body Region [Articolo su rivista]
Smith, Frank W; Boothby, Thomas C; Giovannini, Ilaria; Rebecchi, Lorena; Jockusch, Elizabeth L; Goldstein, Bob

The superphylum Panarthropoda (Arthropoda, Onychophora, and Tardigrada) exhibits a remarkable diversity of segment morphologies, enabling these animals to occupy diverse ecological niches. The molecular identities of these segments are specified by Hox genes and other axis patterning genes during development [1, 2]. Comparisons of molecular segment identities between arthropodandonychophoran species have yielded important insights into the origins and diversification of their body plans [3–9]. However, the relationship of the segments of tardigrades to those of arthropods and onychophorans has remained enigmatic [10, 11], limiting our understanding of early panarthropod body plan diversification. Here, we reveal molecular identities for all of the segments of a tardigrade. Based on our analysis, we conclude that tardigrades have lost a large intermediate region of thebody axis—aregion correspondingto the entire thorax and most of the abdomen of insects— and that they have lost the Hox genes that originally specified this region. Our data suggest that nearly the entire tardigrade body axis is homologous to just the head region of arthropods. Based on our results, we reconstruct a last common ancestor of Panarthropoda that had a relatively elongate body plan like most arthropods and onychophorans, rather than a compact, tardigrade-like body plan. These results demonstrate that the body plan of an animal phylum can originate by the loss of a large part of the body.

2016 - They are among us: the European invasion of the alien brown marmorated stinkbugs Halyomorpha halys (Heteroptera, Pentatomidae) [Poster]
Piemontese, L.; Cesari, Michele; Maistrello, Lara; Giovannini, Ilaria; Dioli, P.; Partsinevelos, G. K.; Rebecchi, Lorena; Guidetti, Roberto

The brown marmorated stinkbug (BMSB), Halyomorpha halys is an invasive alien species native to eastern Asia. Its presence outside the original area of distribution has been recorded for the first time in North America (Canada and U.S.A.) and, more recently, in Europe (Switzerland, France, Hungary, Romania, Austria, Serbia and Italy), where H. halys is spreading rapidly. Other than being a household pest all over its introduced range, this stinkbug is causing great economic losses in the U.S.A and Italian orchards/crops due to its highly polyphagous nature and bivoltinism. Tracing back the pattern of introduction and monitoring the spread of BMSB in the European territory will be useful to implement better pest control strategies. The present study aimed to identify the potential pathways of entry of H. halys in Europe by detecting the genetic diversity of specimens collected all over Italy, and in Switzerland, Romania and Greece. The analyses of 1,175 bp of mitochondrial DNA cytochrome c oxidase I and II genes (cox1 and cox2) of over 200 specimens led to the identification of 12 haplotypes never observed before (10 for cox1 and 2 for cox2). Present data indicate a higher haplotype diversity of European specimens compared with the American ones; instead, the diversity is lower with regard to the Asian samples, except for the cox2 marker. A clear-cut difference in haplotype distribution was found between North and South Europe: Switzerland and France share a similar haplotype pattern, whereas Italian, Hungarian, Romanian and Greek samples are more similar, with the Italian and Greek specimens showing the higher genetic diversity. In Italy, genetic diversity for both markers is higher in Piedmont, Lombardy and Veneto, while in the remaining regions of Northern Italy in which the species is spread, it is fairly low. Haplotype similarity with both Chinese and Korean samples led to hypothesize that the introduction of H. halys in Europe, and in Italy in particular, has occurred by means of multiple events from Asia and that the BMSB is currently expanding its range in the European continent.

2016 - What if the claws are reduced? Morphological and molecular phylogenetic relationships of the genus Haplomacrobiotus May, 1948 (Eutardigrada, Parachela) [Articolo su rivista]
Cesari, Michele; Vecchi, Matteo; Palmer, Aparna; Bertolani, Roberto; Pilato, Giovanni; Rebecchi, Lorena; Guidetti, Roberto

Eutardigrada systematics relies mainly on the morphology of the sclerified structures of the animals. In particular, the main division of Parachela into four superfamilies relies heavily upon claw morphology; however, this character, alone, may be either inadequate or useless for tardigrades with no claws, or when secondary claw branches are reduced or absent, as in the case of species belonging to the genus Haplomacrobiotus. This is a very uncommon genus, so far reported only in North America. The systematic position of this genus has been debated since its description, having first been placed in the family Macrobiotidae (Macrobiotoidea) and then in the family Calohypsibiidae (Hypsibioidea). Currently, the position of the supposedly related genus Hexapodibius is still debated, being attributed to Isohypsibiidae (Isohypsibioidea) or to Calohypsibiidae (Hypsibioidea), i.e. to two different superfamilies. The morphological (light and electron microscopy), chemical (X–ray spectroscopy), and molecular (18S and 28S genes) analyses of a population of Haplomacrobiotus utahensis Pilato & Beasley, 2005 from Snow Canyon State Park (Utah, USA) allowed us to validate the position of this genus within Isohypsibioidea. Our integrated findings placed Haplomacrobiotus in a close relationship with the genus Hexapodibius, and allowed us to erect the new family Hexapodibiidae, comprising the genera Hexapodibius, Parhexapodibius, Haplomacrobiotus, and Haplohexapodibius.

2015 - A pest alien invasion in progress: potential pathways of origin of the brown marmorated stink bug Halyomorpha halys populations in Italy [Articolo su rivista]
Cesari, Michele; Maistrello, Lara; Ganzerli, Francesco; Dioli, Paride; Rebecchi, Lorena; Guidetti, Roberto

he brown marmorated stink bug, Halyomorpha halys, is an agricultural and household pest native of far East. In the last years, it has spread to and established in countries outside its area of origin, most notably in North America (United States and Canada), causing severe economic losses in agricultural crops. Recently, H. halys has been found in Europe (Switzerland, Germany, France, Hungary, and Greece) and since September 2012, it has also been found in Italy. However, the modalities of introduction and spreading of this pest on the Italian territory are unknown. Tracing back the diffusion modes of the species by analyzing the genetic structure and composition of populations in their initial phase of colonization could be useful also in the view to implement better pest control strategies. The present study aimed to identify the potential pathways of entry of H. halys by detecting the genetic diversity of specimens collected from Northern Italy and Canton Ticino (Southern Switzerland). The analyses of 1,175 base pairs of mtDNA cytochrome c oxidase I and II genes (cox1 and cox2) on 42 specimens led to the identification of four combined haplotypes: one, found in Emilia Romagna region, is the same found in China and North America but never observed before in Europe. The other combined haplotypes are new but consistent in part with haplotypes previously found in Switzerland. Present data indicate that the Italian invasion may have occurred from two different pathways, both from Switzerland and from Asia and/or North America.

2015 - Brown marmorated stink bugs are invading Europe: potential pathways of origin of the alien pest populations of Halyomorpha halys (Heteroptera, Pentatomidae) [Abstract in Atti di Convegno]
Cesari, Michele; Maistrello, Lara; Piemontese, Lucia; Dioli, Paride; PARTSINEVELOS Georgios, K.; Rebecchi, Lorena; Guidetti, Roberto

The brown marmorated stink bug, Halyomorpha halys is an agricultural and household pest originating from Asia. In the last years it has become an invasive pest in North America causing severe economic losses to agricultural crops in the United States. Recently, H. halys has been retrieved in Europe (Switzerland, Germany, France, Hungary, and Greece) and, since September 2012, it has also been found in Italy. Tracing back the patterns of introduction and monitoring the spread of H. halys in the Italian territory in its initial phase of colonization will be useful in the view to implement better pest control strategies. The present study aims to identify the potential pathways of entry of H. halys by detecting the genetic diversity of specimens collected in Northern Italy, Southern Switzerland and Greece. The analyses of 1,175 base pairs of mitochondrial DNA cytochrome c oxidase I and II genes (cox1 and cox2) on more than 130 specimens led to the identification of ten haplotypes: one, scored in Italy and Greece, is the same found both in China and North America, while two haplotypes found in Switzerland and Lombardy are shared only with Chinese specimens. The other seven haplotypes are new and present high similarity with Asian haplotypes. Present data show that the introduction of the brown marmorated stink bug in Europe has occurred by means of multiple events, probably both from Asia and North America, and that H. halys is currently expanding its range in the European continent.

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 - Life history traits and reproductive mode of the tardigrade Acutuncus antarcticus under laboratory conditions: strategies to colonize the Antarctic environment [Articolo su rivista]
Altiero, T.; Giovannini, I.; Guidetti, R.; Rebecchi, L.

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 bet-hedging 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 - Phylum Tardigrada [Capitolo/Saggio]
Nelson, Diane R.; Guidetti, Roberto; Rebecchi, Lorena

Anatomy, reprodcution, systematics, taxonony, plylogeny, and dormant state of freshwater tardigrades.

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.

2015 - Superoxide Anion Radical Production in the Tardigrade Paramacrobiotus richtersi, the First Electron Paramagnetic Resonance Spin-Trapping Study [Articolo su rivista]
Savic, Aleksandar G; Guidetti, Roberto; Turi, Ana; Pavicevic, Aleksandra; Giovannini, Ilaria; Rebecchi, Lorena; Mojovic, Milos

Anhydrobiosis is an adaptive strategy that allows withstanding almost complete body water loss. It has been developed independently by many organisms belonging to different evolutionary lines, including tardigrades. The loss of water during anhydrobiotic processes leads to oxidative stress. To date, the metabolism of free radicals in tardigrades remained unclear. We present a method for in vivo monitoring of free radical production in tardigrades, based on electron paramagnetic resonance and spin-trap DEPMPO, which provides simultaneous identification of various spin adducts (i.e., different types of free radicals). The spin trap can be easily absorbed in animals, and tardigrades stay alive during the measurements and during 24-h monitoring after the treatment. The results show that hydrated specimens of the tardigrade Paramacrobiotus richtersi produce the pure superoxide anion radical ((•)O2(-)). This is an unexpected result, as all previously examined animals and plants produce both superoxide anion radical and hydroxyl radical ((•)OH) or exclusively hydroxyl radical.

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 - Desiccation tolerance and production of Reactive Oxygen Species (ROS) in the anhydrobiotic water bear Paramacrobiotus richters [Abstract in Atti di Convegno]
Giovannini, Ilaria; Guidetti, Roberto; Rebecchi, Lorena

The production of Reactive Oxygen Species (ROS) during desiccation processes is documented in bacteria and plants, whereas studies on animals are in practice lacking. In this study we investigated the response to dehydration with respect to the ROS production during the kinetic of the desiccation process and the short and long-time permanence in the anhydrobiotic state. We hypothesized that ROS production, and thus oxidative damage, may be greater in animals maintained desiccated for long-time than in animals maintained dry just for one day. The tardigrade Paramacrobiotus richtersi was used as a target animal. In parallel, oxidative stress was assessed in hydrated P. richtersi as control. Adults were experimentally dehydrated in laboratory using an optimal desiccation protocol to achieve a 100% survival rate of P. richtersi. Animals were maintained desiccated (at 3% RH and 20°C) from 1 to 40 days. Intracellular ROS production during rehydration was evaluated in the “storage cells” (free-floating cells in the tardigrade body cavity), after treatment of the tardigrades with the probe 2,7-dichlorodihydrofluorescein-diacetate (DCFH2-DA). The amount of green fluorescent oxidation product (DCF), which reflects the reaction of the probe with intracellular free radicals, was measure by a laser scanner confocal microscope. The desiccation process does not produce a high amount of ROS, even though previous studies on the same species, P. richtersi, demonstrated an increase of antioxidant enzyme activity in desiccated specimens with respect to hydrated ones. Instead, the long-time permanence in anhydrobiosis (20 days) produces a significant increase of ROS, as evidenced after three and twelve hours from rehydration. The collection of data about the permanence in anhydrobiosis for 40 days is in progress. The high amount of ROS detected in animals maintained dry for long-time, and consequently the oxidative molecular damages, justifies that tardigrade survival decreases with the time spent in a dry state.

2014 - Mopsechiniscus franciscae, a new species of a rare genus of Tardigrada from continental Antarctica [Articolo su rivista]
Guidetti, Roberto; Rebecchi, Lorena; Cesari, Michele; S. J., Mcinnes

Despite the importance and regular occurrence of tardigrades in the Antarctic terrestrial ecosystem, taxonomic studies of, in particular, continental Antarctica species have advanced very slowly. During a large survey to study tardigrade biodiversity along the Victoria Land coastal line, a new species was found belonging to the rare heterotardigrade genus Mopsechiniscus. The new species Mopsechiniscus franciscae is described using an integrative taxonomy approach, combining morphological description (with light and electron microscopy techniques) and molecular characterisation (analysing portions of the 18S and 28S genes). The new species differed from other congeners by clear morphological characters related to shape and sculpture of cuticular plates, presence of papillae on legs, and length and number of body filaments. The results of the combined (18S+28S) phylogenetic analyses (Bayesian and Maximum Likelihood) on Echiniscoidea indicate two main lineages: one incorporating the genus Echiniscoides (Echiniscoididae) and the other the current data on Echiniscidae and Oreellidae genera. Although the resolution of relationships within the latter line is not clear, there is a well defined evolutionary line for Mopsechiniscus. The addition of continental Antarctic M. franciscae sp. nov. to the genus broadened the distributional range of Mopsechiniscus southwards and supported the hypothesis that the genus represents a Gondwanan faunal element. Our report of a new Antarctic species, belonging to this rare heterotardigrade genus, increases our knowledge of the underreported terrestrial meiofaunal communities within continental Antarctica.

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 invasion of the new alien species Halyomorpha halys (Hemiptera, Pentatomidae): genetic diversity and possible areas of origin of the populations identified in Italy and Canton Ticino. [Abstract in Rivista]
Guidetti, Roberto; Cesari, Michele; Ganzerli, F.; Dioli, P.; Rebecchi, Lorena; Maistrello, Lara

The invasion of the new alien species Halyomorpha halys (Hemiptera, Pentatomidae): genetic diversity and possible areas of origin of the populations identified in Italy and Canton Ticino.

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.

2014 - The value of the integrative approach in tardigrade taxonomy: the case study of an Antarctic eutardigrade. [Abstract in Rivista]
Bertolani, Roberto; Cesari, Michele; Vecchi, Matteo; Rebecchi, Lorena; Guidetti, Roberto

The use of the molecular approach in tardigrades has recently widespread for identifying taxa and evolutionary lineages. Firstly, it has been utilized for identifying the phylum position within Ecdysozoa and then to recognize both its evolutionary lineages and species. As regards phylogeny, in several cases a good correspondence between molecular and morphological data was found, but in others results were contrasting. For example, the Eutardigrada genera Hypsibius and Isohypsibius, which have two different types of claws sharing asymmetry with respect to the median plane of the leg, were considered belonging to the same subfamily, and 50 years ago subgenera of the same genus, Hypsibius. Molecular data have pointed out that these two genera belong to different superfamilies (Hypsibioidea and Isohypsibioidea). Other genera were attributed to one or the other superfamily according to the morphology of their claws, attributions always confirmed when molecular support was obtained. In Antarctica we found tardigrades with claws attributable to Ramajendas (Isohypsibioidea, Isohypsibiidae) for the particular length of the main branch of the external claw and its weak connection to the basal tract. Ramajendas is only from Antarctica and surrounding austral areas. We also found other specimens, belonging to Ramazzottius and Hebesuncus (Hypsibioidea, Ramazzottiidae). This material allowed us to carry out an integrative approach, obtaining both molecular data (18S and 28S genes) and new morphological information. Both Bayesian and maximum likelihood cladograms clearly placed the specimens with putative Ramajendas claws together with Hebesuncus and Ramazzottius, within the Ramazzottiidae family. New morphological data on the specimens with putative Ramajendas claws evidenced that, like in Ramazzottius, there are two elliptical structures on the head and the eggs are ornamented and laid freely (on the contrary, in all Isohypsibioidea the eggs are smooth shelled and laid within the exuvium). Therefore, both approaches evidence that the specimens do not belong to Ramajendas (and consequently not to Isohypsibioidea), as previously supposed, even though they share their particular shape of the external claw with Ramajendas, evidently due to adaptive convergence. They belong to Hypsibioidea and in particular to a new species of Ramazzottius. These results confirm the importance of the integrative approach in dealing with Tardigrada identification and phylogeny.

2013 - A DNA barcoding approach in the study of tardigrades [Articolo su rivista]
Cesari, Michele; Guidetti, Roberto; Rebecchi, Lorena; Giovannini, Ilaria; Bertolani, Roberto

DNA barcoding is a technique proposed by Hebert and coworkers in 2003 for discriminating species through analysis of a single gene barcode locus. It aims to obtain a better taxonomic resolution than that achieved through morphological studies, and to avoid the decline in taxonomic knowledge. Today DNA barcoding is a global enterprise, and the implementation of the idea has seen a rapid rise (more than 1900 papers published to date on different organisms). Nonetheless, controversy still arises regarding barcoding and taxonomy. It is important to note that DNA barcoding does not focus on building a tree-of-life or on doing DNA taxonomy, even though sometimes it has been used for these purposes. DNA barcoding rather focuses on producing a universal molecular identification key based on strong taxonomic knowledge that should be included in the barcode reference library. In the phylum Tardigrada, DNA barcoding represents a recent approach to species identification and to help in solving taxonomic problems, especially considering the diminutive size of these animals and the paucity of morphological characters useful for taxonomy. In the framework of the MoDNA Project (Morphology and DNA), carried out by our research group in collaboration with several colleagues, we are combining the study of a fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1) with morphological data, in a wide sense (cuticular structures, chromosomes, data on sex ratio and reproduction), to form an integrative taxonomy approach for tardigrade species identification. We believe that without verified reference sequences from voucher specimens that have been authenticated by qualified taxonomists, there is no reliable library for newly generated sequences with which to be compared. Methods and protocols for standardized results are focused on obtaining tight correspondence between tardigrade morphology (and egg shell morphology, when useful), possibly both light and scanning electron microscopy images, and molecular sequence. This approach is particularly useful in describing new species, and important when applied on material collected in species type localities. Results using this approach are presented, primarily focusing on a number of species from the so-called “Macrobiotus hufelandi group”.

2013 - Comparative analysis of the tardigrade feeding apparatus: adaptive convergence and evolutionary pattern of the piercing stylet system [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena

A thorough analysis of the cuticular parts of tardigrade feeding apparatuses was performed in order to provide a more complete understanding of their evolution and their potential homologies with other animal phyla (e.g. Cycloneuralia and Arthropoda). The buccal- pharyngeal apparatuses of eight species belonging to both Eutardigrada and Heterotardigrada were studied using light and scanning electron microscopy. This study supports and completes a previous study on the relationships between form and function in the buccalpharyngeal apparatus of eutardigrades. The common sclerified structures of the tardigrade buccal-pharyngeal apparatus are: a buccal ring connected to a straight buccal tube, a buccal crown, longitudinal thickenings within the pharynx, and a stylet system composed of piercing stylets within stylet coats, and stylet supports. Specifically, heterotardigrades (Echiniscoidea) have a narrow buccal tube; long piercing stylets, each with a longitudinal groove, that cross one another before exiting the mouth; pharyngeal bars and secondary longitudinal thickenings within the pharynx. In contrast, eutardigrades have stylets which are shorter than the buccal tube; Parachela have pharyngeal apophyses and placoids within the pharynx, while Apochela lack a buccal crown and cuticular thickenings within the pharynx, the buccal tube is very wide, and the short stylets are associated with triangular-shaped stylet supports. In both classes, when the piercing stylet tips emerge from the mouth to pierce food, the buccal tube opening is almost completely obstructed, which may hinder food uptake. In heterotardigrades, the crossing of the piercing stylets may further decrease food uptake, however this disadvantage may have been reduced in echiniscids by the evolution of a long buccal tube and long stylets able to run more parallel to the buccal tube. In contrast, eutardigrades evolved different strategies. In the order Apochela and in several Itaquasconinae (Parachela), the buccal tube is wide, the stylets are short and run parallel to the tube without crossing. In other Parachela, the piercing stylets do not cross one another because they are curved. Further, the development of an anterior bend in the buccal tube (e.g. in Doryphoribius and Macrobiotoidea) may allow the shift of the stylet sheaths to a more ventral position so that a wide portion of the mouth is free during the piercing stylet movements. The possible convergent evolution of several structures of the buccal-pharyngeal apparatus (e.g. ventral lamina, pharyngeal tube, wide buccal tube without buccal crown, buccal lamellae) was analysed and discussed.

2013 - Diversità genetica e distribuzione geografica in una specie pan antartica: Acutuncus antarcticus (Tardigrada, Hypsibiidae [Abstract in Rivista]
Cesari, Michele; Mcinnes, S.; Bertolani, Roberto; Mori, L.; Rebecchi, Lorena; Guidetti, Roberto

L’origine della fauna continentale antartica è oggetto di ampia discussione. La distribuzione dei vari taxa potrebbe essere il risultato di una dispersione in atto (recolonization hypothesis), oppure di una distribuzione Gondwaniana (glacial refugia hypothesis). Entrambe le ipotesi mancano però di conferme, considerando anche le scarse conoscenze sulla fauna invertebrata antartica. Per trovare prove a favore di una o dell’altra ipotesi è stata quindi iniziata l’analisi della diversità genetica e della distribuzione dei tardigradi, uno dei taxa più rappresentati nella fauna antartica continentale. Sono stati analizzati esemplari della specie endemica Acutuncus antarcticus (Eutardigrada, Hypsibiidae), campionati in sedimenti di pozze permanenti o semipermanenti provenienti da 7 località poste lungo un transetto nord-sud in Terra Vittoria. I due punti di campionamento più vicini tra loro distavano circa 23 km, mentre i più lontani circa 622 km. Per 65 esemplari sono state ottenute le sequenze dei geni 18S e cox1, che sono poi state analizzate insieme alle corrispondenti sequenze disponibili in GenBank e BOLD. E’ stata calcolata la diversità genetica e aplotipica tra individui (p-distance, Kimura 2-parametri) e tra popolazioni (diversità genetica di Nei, FST, numero di migranti) e costruita una rete di aplotipi. Su alcune popolazioni è stata inoltre effettuata l’analisi cariologica e valutata la sex ratio. Tutti gli individui (analizzati ex novo o disponibili nelle banche dati) sono risultati appartenere alla stessa specie: essi presentavano la stessa sequenza per il gene 18S e soprattutto una distanza genetica per il gene cox1 minore del 5%. La maggior parte delle popolazioni presentava più aplotipi, con l’eccezione di quelle situate nella parte più a nord del transetto, che sono risultate omogenee. Alcuni aplotipi risultavano presenti in diverse località anche distanti tra loro. Le popolazioni, sempre formate da sole femmine (2n= 12/14), sono geneticamente differenziate, ad eccezione di quelle situate nella parte centrale del transetto. Acutuncus antarcticus risulta essere una specie panantartica molto diffusa, in ambiente dulciacquicolo. Le popolazioni con maggiore variabilità genetica sono in concomitanza con l’hotspot di biodiversità individuato sulle coste di Terra Vittoria, che si ipotizza sia servito da glacial refugium durante gli eventi di glaciazione che caratterizzano il continente.

2013 - Dry and survive: the role of antioxidant defences in anhydrobiotic organisms [Articolo su rivista]
Rebecchi, Lorena

Although evolution of life has turned oxygen into a vital compound for aerobic organisms, this element can also have deleterious effects on living systems being involved in the production of oxidative stress. This is a process resulting from an imbalance between excessive production of reactive oxygen species (ROS) and limited action of antioxidant defenses. It is a particularly harmful health risk factor, involved in the development of several chronic human pathologies and believed to play a major role in the ageing process. Consequently aerobic metabolism needs a stringent control of ROS. Water too is essential for life, but some organisms widespread throughout nature have the ability to survive complete desiccation by entering in anhydrobiotic state. The loss of water involves important biological processes such as changes in metabolism, modifications of cell membranes, and production of ROS. In anhydrobiotic state, high temperatures, high humidity, light exposure and high oxygen partial pressure negatively affect organism survival and directly influence the time required by them to recover active life after a period of desiccation. These abiotic factors induce damages that are accumulated in proportion to the time spent in the desiccated state, leading to organism death. Oxidative stress seems to be one of the most deleterious damages due to water depletion, therefore anhydrobiosis needs a stringent control of ROS production too. Anhydrobiotes seem to apply two main strategies to cope with the danger of oxygen toxicity, namely an increasing efficiency of antioxidant defences and a metabolic control of both energy-production and energy-consuming processes. Experimental studies produced evidence that antioxidant defences such as ROS scavenging enzymes (e.g. peroxidases, catalases, superoxide dismutase, glutathione peroxidases) and other molecules (e.g. glutathione, carotenoids, vitamins C and E) represent a key group of molecules required for desiccation tolerance in tardigrades. The action of these molecules emphasises the need for redox balancing in anhydrobiotic organisms including tardigrades and sleeping chironomid larvae.

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 - Essiccamento e produzione di specie reattive dell’ossigeno (ROS) in tardigradi anidrobionti [Abstract in Rivista]
Giovannini, Ilaria; Guidetti, Roberto; Rebecchi, Lorena

Anche se l’acqua è essenziale per la vita, organismi di linee evolutive diverse hanno sviluppato la capacità di tollerare un essiccamento estremo entrando in un particolare stato fisiologico reversibile, detto anidrobiosi. Con questo adattamento, rotiferi bdelloidei, nematodi e tardigradi perdono fino al 97% dell’acqua corporea, con sospensione del metabolismo e cambiamenti nell’organizzazione e composizione molecolare delle membrane cellulari. Nei tardigradi essiccati, la sopravvivenza a lungo termine è inversamente proporzionale a temperatura, umidità relativa dell’aria e pressione parziale di ossigeno, fattori abiotici che contribuiscono a danneggiare le molecole biologiche. Una delle principali cause di danno durante l’anidrobiosi sembra essere lo stress ossidativo, dovuto al disequilibrio fra l’eccessiva produzione di specie reattive dell’ossigeno (ROS) e la limitata attività degli antiossidanti. Tuttavia, la produzione di ROS durante l’essiccamento è ben documentata solo in pochi organismi, soprattutto autotrofi, mentre mancano praticamente dati sugli animali anidrobionti e, soprattutto, sull’effettivo accumulo di ROS durante la permanenza in anidrobiosi per lunghi periodi di tempo. È stata quindi valutata la produzione di ROS nell’eutardigrado anidrobionte Paramacrobiotus richtersi, analizzando animali essiccati sperimentalmente in laboratorio [4 h a 18°C e 80% di umidità relativa dell’aria (RH); 4 h a 18°C e 50% RH; 12 h in gel di silice] e mantenuti essiccati per vari periodi di tempo (da 1 a 30 giorni) o sottoposti a temperature elevate (37°C e 60°C). Come controllo sono stati utilizzati animali mantenuti idratati. La produzione di ROS è stata valutata nei globuli cavitari, cellule libere nella cavità corporea dei tardigradi, dopo trattamento degli animali con il marcatore 2,7 diclorodiidrofluoresceina diacetato (DCFH2-DA) e successiva rilevazione della quantità del prodotto di ossidazione fluorescente 2,7 diclorofluoresceina (DFC), mediante microscopia confocale a scansione laser. I primi dati indicano che in P. richtersi l’entrata in anidrobiosi di per sé non determina la produzione di elevate quantità di ROS, sebbene sia stato dimostrato un incremento dell’attività degli enzimi antiossidanti negli esemplari essiccati rispetto a quelli idratati. La produzione di ROS sembra aumentare quando gli animali essiccati sono mantenuti in anidrobiosi per lunghi periodi di tempo ed esposti ad alti valori di temperatura.

2013 - Integrative taxonomy allows the identification of synonymous species and the erection of a new genus of Echiniscidae (Tardigrada, Heterotardigrada) [Articolo su rivista]
Filipe, Vicente; Paulo, Fontoura; Cesari, Michele; Rebecchi, Lorena; Guidetti, Roberto; Artur, Serrano; Bertolani, Roberto

The taxonomy of tardigrades is challenging as these animals demonstrate a limited number of useful morphological characters, therefore several species descriptions are supported by only minor differences. For example, Echiniscus oihonnae and Echiniscus multispinosus are separated exclusively by the absence or presence of dorsal spines at position Bd. Doubts were raised on the validity of these two species, which were often sampled together. Using an integrative approach, based on genetic and morphological investigations, we studied two new Portuguese populations, and compared these with archived collections. We have determined that the two species must be considered synonymous with Echiniscus oihonnae the senior synonym. Our study showed generally low genetic distances of cox1 gene (with a maximum of 4.1%), with specimens displaying both morphologies sharing the same haplotype, and revealed character Bd to be variable. Additionally, a more detailed morphological and phylogenetic study based on the 18S gene uncovered a new evolutionary line within the Echiniscidae, which justified the erection of Diploechiniscus gen. nov. The new genus is in a sister group relationship with Echiniscus and is, for the moment, composed of a single species.

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 - Proceedings of the 12th International Symposium on tardigrada [Curatela]
Rebecchi, Lorena; D., R: Nelson; Bertolani, Roberto; P., Fontoura

Proceedings of 12th International Symposium on Tardigrada. 23-26 July 2012 Vila Nova de Gaia Portugal

2013 - Somatic muscolature of Tardigrada: phylogenetic signal and metameric patterns [Articolo su rivista]
Marchioro, Trevor; Rebecchi, Lorena; Cesari, Michele; Hansen, J. G.; Viotti, Giulia; Guidetti, Roberto

Although studies describing molecular-based phylogenies within tardigrades are now frequently being published, this is not the case for studies combining molecular and morphological characters. Tardigrade phylogeny is still based, from a morphological point of view, almost exclusively on chitinous structures and little attention has been given to detecting and using novel morphological data. Consequently, we analysed the musculature of seven tardigrade species belonging to the main phyletic lines by confocal laser scanning microscopy and compared these morphological results with new molecular analyses (18S+28S rRNA genes). Finally, we analysed all the data with a total evidence approach. A consilience in the phylogenetic relationships among orders and superfamilies of tardigrades was obtained among the evolutionary trees obtained from morphological, molecular and total evidence approaches. Comparative analysis on the musculature allowed the identification of serial homologies and repeated metameric patterns along the longitudinal animal body axis. A phenomenon of mosaic evolution was detected in musculature anatomy, as dorsal musculature was found to be highly modified with respect to the other body muscle groups, probably related to the evolution of dorsal cuticular plates. An understanding of tardigrade musculature anatomy will give fundamental information to understand the evolution of segmental pattern within Panarthropoda.

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

2013 - Thermal stress induces HSP70 proteins synthesis in larvae of the cold stream non-biting midge Diamesa cinerella Meigen. [Articolo su rivista]
Lencioni, Valeria; Paola, Bernabò; Cesari, Michele; Rebecchi, Lorena

Laboratory experiments on the cold stenothermal midge Diamesa cinerella (Diptera, Chironomidae) were performed to study the relationship between temperature and heat shock proteins (HSP70) expression at translational level (Western blotting). Thermotolerance of IV instar larvae collected in nature at 1.5-4.3 °C were analyzed through short-term (1 h at ten different temperatures from 26 °C and 35°C) and long-term (2-14 h at 26°C and 2-4 h at 32°C) heat shocks. A high thermotolerance was detected (LT50 = 30.9-32.8 °C and LT100 =34.0-37.8 °C). However, survival decreased consistently with increasing exposure time, especially at higher temperature (LTime50 = 7.64 h at 26°C and LTime50 = 1.73 h at 32°C). The relationship between such heat resistance and HSP70 expression appeared evident since a relationship between HSP70 level and survival rate was generally found. A Heat Shock Response was consistent only in the summer larvae, but the highest levels of HSP70 both in the control and in heat stressed larvae were found in the winter population. The high resistance to heat found in the winter population (LT50= 32.5 ± 0.5 °C) seems to be given more by constitutive high levels of HSP70 proteins than by HSR induction. Altogether these results stressed how the HSP70 protein family confers resistance against cold, being detected under natural conditions in control larvae collected in all seasons, and against heat under shocks. These results give new insights into possible responses to climate changes in freshwater insects within the context of global warming.

2012 - A DNA barcoding approach in the study of tardigrades. [Abstract in Atti di Convegno]
Cesari, Michele; Rebecchi, Lorena; Guidetti, Roberto; Giovannini, Ilaria; Mori, L.; Bertolani, Roberto

DNA barcoding is a technique proposed by Hebert and coworkers in 2003 and it aims to discriminate biological entities through analysis of a single gene barcode locus. The DNA barcoding system promised a better taxonomic resolution than that achieved through morphological studies, with a partial solution to the decline in taxonomic knowledge. Today DNA barcoding is a global enterprise, and the implementation of the idea has seen a rapid rise (more than 450 papers published to date on different organisms). Nonetheless, controversy still arises regarding barcoding and taxonomy. It is important to note that DNA barcoding does not focus on building a tree-of-life or on doing DNA taxonomy, even though sometimes it has been used for these purposes. DNA barcoding rather focuses on producing a universal molecular identification key based on strong taxonomic knowledge that should be included in the barcode reference library. In Phylum Tardigrada, DNA barcoding represents a recent approach to species identification and for helping to solve taxonomic problems, especially considering the diminutive size of these animals and the paucity of morphological characters useful for taxonomy. In the framework of the MoDNA Project (Morphology and DNA), carried out by our research group in collaboration with several colleagues, we are combining the study of a fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1) with morphological data, in a wide sense, to form an integrative taxonomy of tardigrades. Building of a database of reference sequences is of paramount importance for a correct application of DNA barcoding in tardigrades. Without verified reference sequences from voucher specimens that have been authenticated by qualified taxonomists, there is no reliable library for newly generated sequences with which to be compared. Methods and protocols for standardized results are focused on obtaining tight correspondence between molecular sequence and animal morphology, possibly both LM and SEM images (and egg shell morphology, when useful). This approach is particularly useful in describing new species, and important when applied on material collected in type localities. Results using this approach are presented, focusing primarily on a number of species from the so-called “Macrobiotus hufelandi group”.

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 - Comparative analysis and phylogenetic implications of tardigrade musculature architectures. [Abstract in Atti di Convegno]
Marchioro, Trevor; Rebecchi, Lorena; Hansen, J. Gulberg; Bertolani, Roberto; Guidetti, Roberto

Most knowledge on tardigrade musculature architecture dates back to the end of XIX century, and the beginning of XX century. It has been only in the last five years that a great deal of new information on tardigrade musculature system has become available, mainly thanks to the use of rhodamine-phalloidin staining of F-actin in combination with three-dimensional microscopical techniques such as confocal laser scanning microscopy (CLSM). In spite of all these information, only few and fragmentary evolutionary considerations on tardigrade musculature system have been done. This is probably due to the relatively low number of analyzed taxa, and to the difficulty in the comparisons of data that often have been obtained with different degree of accuracy, and are presented using different terminologies. In this study we increased the number of analyzed species, by studying CLSM the musculature architectures of 7 species representative of most tardigrade higher taxa: the heterotardigrades Batillipes bullacaudatus (Arthrotardigrada), and Echiniscus testudo (Echiniscoidea), and the eutardigrades Paramacrobiotus richtersi, Dactylobiotus parthenogeneticus (Parachela, Macrobiotoidea), Bertolanius volubilis (Parachela, Eohypsibioidea), Acutuncus antarcticus (Parachela, Hypsibioidea) and Milnesium tardigradum (Apochela, Milnesiidae). We were able to define all the muscular fibers associated with the body movement. The number of fibers and their organization changed among taxa, with heterotardigrades being the least complex. Muscular fibers have been schematically organized into three systems: dorsal, lateral, and ventral. The ventral system was the most conservative, showing a clear metameric pattern and only few differences among taxa, while the lateral system was the most derived and so precise homologies were not always well defined. Using these new morphological information and literature data on Halobiotus crispae (Parachela, Isohypsibioidea), it was possible to analyse the phylogenetic signal of the musculature system. Two matrixes were constructed: a morphological matrix of 94 characters based on musculature data, and a matrix for a total evidence analysis combining the previous data with molecular data (18S, 28S). Both matrixes have been analyzed in Bayesian and parsimony frameworks. The phylogenetic trees, obtained by both analyses using both matrixes, differ only for the position of Eohypsibioidea. Heterotardigrade taxa were the sister group of all Eutardigrada, within this last class, Apochela and Parachela were sister taxa; among parachelan superfamilies the Isohypsibioidea was the most basal, the Macrobiotoidea the most derived, while the Eohypsibioidea changed position according to the analyses. Our data demonstrated that musculature architecture can be used for phylogenetic purposes, but it is only applicable at higher taxonomic levels. Indeed, the musculature of Paramacrobiotus and Dactylobiotus, although belonging to different families of Macrobiotoidea, showed the same muscular organization.

2012 - Dating tardigrade evolution and early terrestrialization events. [Abstract in Atti di Convegno]
Marchioro, Trevor; Rota Stabelli, O.; Rebecchi, Lorena; Guidetti, Roberto; Pisani, D.

The tempo and mode of cryptobiosis evolution within tardigrades are still unknown, but it is clear that comprehending this process is the key to better understand evolutionary history and ecology of this phylum, and the process of animal terrestrialization, given the ubiquitous continental distribution of tardigrades. Our study was mainly aimed at timing tardigrade radiation and key events in tardigrade evolution. Given the existence of two lineages of continental tardigrades, i.e. Eutardigrada and Echiniscidae, we decided to implement a relaxed molecular clock based approach to attempt to derive a minimal time interval for tardigrade terrestrialization. Data from new and Genbank partial 18S and 28S rDNA sequences of 41 specimens, belonging to 31 species from 26 genera, representing all known tardigrade orders and superfamilies, were acquired. Phylogenetic inference was achieved using Bayesian mixture models that have allowed the most accurate estimates of evolutionary rates. The following molecular clock analyses were then based on 3 tardigrade calibration points derived from fossil records and improved by the addition of 5 new calibrations points pertaining to Arthropoda, Priapulida and Kinoryncha, emerged from paleontological studies. To clarify the evolutionary history of cryptobiosis, and to evaluate whether its origin might have played a role in the process of tardigrade terrestrialization, a maximum-likelihood based ancestral character state reconstruction was used. Molecular phylogeny analyses yielded a robustly supported and well-resolved evolutionary tree for all considered tardigrade taxa. Our results suggest the origin of the tardigrade stem group in the Ediacaran age (~620 Million years ago -Mya), with the two major extant tardigrade lineages (Eutardigrada and Heterotardigrada) split quite recently ~443 Mya. Ancestral character state reconstruction indicated a probability of ~30% for cryptobiosis to have been present in the last common ancestor of Heterotardigrada and Eutardigrada. Hence, it is most likely that this adaptive trait evolved twice independently within this phylum. Cryptobiosis most likely emerged in the time interval between 443 and 359 Mya, in the stem Eutardigrada lineage that existed for ~84 My. This is in accordance with available fossil evidence suggesting terrestrial ecosystems first flourished in the Silurian. The second independent origin (within Heterotardigrada) was within the Echiniscoidea clade and can be dated to an interval included between 238 and 141 Mya (Middle Triassic to early Cretaceous). In both cases, existence of a link between cryptobiosis and terrestrialization was clear. All the eutardigrade superfamilies originated within a short-time span of ~257-200 Mya, suggesting an early Mesozoic eutardigrade radiation. Moreover, within each parachelan superfamily a great mid Jurassic to Early Cretaceous process of diversification was recorded.

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 - Dry and survive: the role of antioxidant metabolism in anhydrobiotic organisms. [Abstract in Atti di Convegno]
Rebecchi, Lorena

Although evolution of life has turned oxygen into a vital compound for most organisms, this element can also have deleterious effects on living systems. Oxidative stress is a process resulting from an imbalance between excessive production of reactive oxygen species (ROS) and limited action of antioxidant defenses. It is a particularly harmful health risk factor, common to the development of several chronic human pathologies (e.g. cancer, Alzheimer’s and Parkinson’s) and believed to play a major role in the ageing process. Thus antioxidant protection is essential for survival under an aerobic environment. Water too is essential for life, but some organisms have the ability to survive extreme desiccation by entering into a state of suspended animation called anhydrobiosis. These organisms are widespread throughout nature, including bacteria, protists, yeasts, plants and animals. The loss of water involves important biological processes such as changes in metabolism, alterations of cell membranes, and production of oxidative stress. Therefore, the maintenance of life in the absence of water requires a complex set of mechanisms working in close coordination, such as the accumulation of bioprotectant molecules, the activation of molecular repair mechanisms and of antioxidant and molecular chaperone systems. Oxidative stress seems to be one of the most deleterious effects of water depletion, since the susceptibility to oxidative damage may increase with dehydration. Anhydrobiotes seem to apply two main strategies to cope with the danger of oxygen toxicity, namely an increasing efficiency of antioxidant defences and a metabolic control of both energy-production and energy-consuming processes. Tardigrades are here presented as model system to evaluate the effective damages induced by an increase of ROS production during desiccation and to understand the role of antioxidant systems to ensure survival of living beings when in the anhydrobiotic state. Even though desiccation does not seem to have an effect on tardigrade longevity, damages are accumulated in proportion to the time spent in the desiccated state, leading to animal death. High temperatures, high humidity and high oxygen partial pressure are all factors that negatively affect tardigrade survival during long-term anhydrobiosis since they are involved in the production of oxidative stress. These abiotic conditions also directly influence the time required by animals to recover active life after a period of desiccation. Experimental studies produced evidence that enzymes (e.g. peroxidases, catalases, superoxide dismutase) and antioxidants (e.g. glutathione and carotenoids) represent a key group of molecules required for desiccation tolerance in tardigrades. The action of these molecules emphasises the need for redox balancing in anhydrobiotic tardigrades.

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 - Morphology, DNA barcoding and phylogeny of Macrobiotus persimilis and Macrobiotus polonicus. [Abstract in Atti di Convegno]
Bertolani, Roberto; Cesari, Michele; Lisi, O.; Rebecchi, Lorena; Giovannini, Ilaria; Pilato, G.

Macrobiotus polonicus and Macrobiotus persimilis are two species of the “Macrobiotus hufelandi group” which are distinguished one from the other for the presence in M. polonicus of one lateral gibbosity on each hind leg, the size of cuticular pores (smaller and less evident in M. polonicus), the buccal tube size (larger in M. persimilis) and the egg processes (larger terminal discs and with more evident indentation in M. polonicus). However, the two taxa share peculiar characteristics: similar values of the pt index relative to the insertion point of the stylet supports on the buccal tube, first macroplacoid clearly longer than the second and with a central constriction, lunules of the hind legs clearly larger than those of the first three pairs and with an indented margin, egg shell smooth or with faint dots but without a reticular design. Therefore, it could be hypothesized that they belong to a single peculiar group, namely the “Macrobiotus polonicus/persimilis group”. In order to verify this hypothesis, a morphological and DNA barcoding analysis (cox1) was carried out on tardigrades attributable to these two taxa and coming from five different localities in Italy and France. This study revealed differences, both morphological and molecular, between some populations apparently belonging to the same species, which in some cases turned out to be cryptic species. A phylogenetic analysis using rDNA 18S was performed in order to verify the relationships among the species of this group and more in general among those of the “M. hufelandi group”. The DNA barcoding analysis revealed a high divergence, with very high values of genetic distance among some populations (more than 18%). In one case (Enna, Sicily) at least two different and very distinct entities are present together. The morphological and morphometrical investigation, also based on type material, confirms this variety, showing that at least four species related to M. polonicus and three related to M. persimilis should be distinct. In several cases the relationship between morphology and cox1 sequence has been ascertained. The dendrogram computed on 18S showed that there are two different sequences pertaining to the “M. polonicus/persimilis group” clustered in a single evolutionary line inside the main M. hufelandi cluster.

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 - An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens [Articolo su rivista]
Cesari, Michele; Giovannini, Ilaria; Bertolani, Roberto; Rebecchi, Lorena

We have in recent papers revealed that an integrative taxonomy approach helps to solve taxonomic problems in tardigrades. However, whole tardigrades are required for DNA work, which leaves no hologenophore voucher specimens with adult morphology. Using a novel methodology for the Tardigrada, we introduce the practice of collecting high quality maximum magnification light microscopy images of recently thawed animals to act as hologenophore voucher specimens of animals later used for DNA barcode sequencing. Within the framework of a DNA barcoding project on tardigrades, we collected a moss sample from the type locality of Macrobiotus terminalis Bertolani & Rebecchi, 1993 (Castelsantangelo, Central Apennines, Italy), a species of the “Macrobiotus hufelandi group”. Within the moss sample we found several animals and eggs with a morphology that corresponded to the original description of M. terminalis, while others were attributable to Macrobiotus macrocalix Bertolani & Rebecchi, 1993. In this study, molecular (cox1 mtDNA) analyses demonstrated no intraspecific variability in M. terminalis from the type locality but very large interspecific differences when compared with M. macrocalix and GenBank data for other species within the M. “hufelandi group”. There was also a large difference between our M. terminalis sequences and the GenBank data of a specimen attributed to the same species. The GenBank sequence originated from a population in the Northern Apennines, whose morphology appeared to be like that of the specimens of the locus typicus. This confirmed the importance in utilising material from the type locality for linking molecular data to the species’ morphological characters. Our paper underlines the importance of an integrative taxonomy in species diagnoses and demonstrates a scenario where morphological observations alone are not always sufficient. Lastly, this work adds reliable information to the sequence reference library that provides a useful building block for further studies on similar and related tardigrade taxa.

2011 - DNA barcoding and integrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the first tardigrade species to be described, and some related species. [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena; Giovannini, Ilaria; Cesari, Michele

Within the framework of a DNA barcoding project on tardigrade species, a study was carried out on Macrobiotus hufelandi C.A.S. Schultze 1834, the first formally described tardigrade species. We used samples collected from the type locality and additional material from other European sites containing species of the “M. hufelandi group”. The study was performed by integrating morphological, karyological and molecular (mt-DNA cox1) information and comparing these data with morphological data from the type material. Several species from this group were found in the type locality of M. hufelandi (near Freiburg, Black Forest, Germany) and these were all barcoded. One was M. hufelandi, the other two were: Macrobiotus sandrae Bertolani & Rebecchi 1993 (originally described from the same locality), and Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari in press (type locality Andalo, Italy), all with interspecific genetic distances of more than 19%. A fourth cryptic species, which had the same morphology as M. hufelandi but a genetic distance of 6.7%, was not described as a new taxon but named M. cf. hufelandi sp.1 for this study. Macrobiotus sandrae and M. vladimiri were also present (and barcoded) in Italy (Alps). Additional individuals (animals and eggs) were also found, and barcoded, in Italy (Apennines) and Switzerland that belonged to the haplogroup Macrobiotus cf. hufelandi sp. 1. These data together with other recent studies on tardigrade DNA barcoding represent a starting point for further studies on tardigrade biogeography, phylogeography and diversity.

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 - Filogenesi e integrative taxonomy nei Macrobiotus del “gruppo hufelandi” (Tardigrada, Eutardigrada) [Abstract in Atti di Convegno]
Cesari, Michele; Rebecchi, Lorena; Giovannini, Ilaria; Pilato, G.; Lisi, O.; Vicente, F.; Kiosya, Y.; Bertolani, Roberto

Analiisi della filogenesi dei Macriobiotus del gruppo hufelandi medainte un approccio integrativo

2011 - MicroRNA and phylogenomics congruently resolve the phylogenetic relationships of the Tardigrada within Ecdysozoa. [Abstract in Atti di Convegno]
Pisani, D.; Campbell, L.; Rota Stabelli, O.; Marchioro, Trevor; Longhorn, S.; Telford, M.; Philippe, H.; Edgecombe, G.; Rebecchi, Lorena; Peterson, K. J.

MicroRNA and phylogenomics congruently resolve the phylogenetic relationships of the Tardigrada within Ecdysozoa

2011 - MicroRNAs and phylogenomics resolve the relationships of Tardigrada, and suggest the velvet worms are the sister group of Arthropoda [Articolo su rivista]
L. I., Campbell; O., Rota Stabelli; G. E., Edgecombe; Marchioro, Trevor; S. J., Longhorn; H., Philippe; M. J., Telford; Rebecchi, Lorena; K. J., Peterson; D., Pisani

Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g. spiders, insects and their allies) in a monophyletic group of invertebrates with walking appendages: the Panarthropoda. Molecular data, however, do not generally support the inclusion of tardigrades within the Panarthropoda, placing them closer to Nematoda (roundworms) instead. Here we present results from the analyses of two independent genomic data sets (expressed sequence tags – ESTs, and microRNAs), congruently resolving the phylogenetic relationships of Tardigrada. Our EST analyses significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations – comparisons of model fit, signal dissection, and taxonomic sampling– we were able to show that support for a Nematoda plus Tardigrada group derives from the phylogenetic artefact of long branch attraction – LBA. Our small RNA libraries – including the first data for tardigrades and onychophorans – fully support our EST results: no microRNAs were found to link Tardigrada and Nematoda, while all panarthropods were found to share one microRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second microRNA (miR-305). To corroborate our results we have applied a methodology similar to that used for the EST dataset to a ribosomal RNA (rRNA) dataset, reaching again the same conclusions. Our study confirming the monophyly of the legged invertebrates, shows that past support for tardigrades plus nematodes was due to LBA, and suggest that the velvet worms are the most likely sister group of Arthropoda.

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 - Taxonomy and biogeography of tardigrades using an integrated approach: new results on species of the Macrobiotus hufelandi group [Articolo su rivista]
Bertolani, Roberto; V. I., Biserov; Rebecchi, Lorena; Cesari, Michele

This study reconsiders a tardigrade population previously studied and tentatively attributed to Macrobiotus cf. terminalis by Bertolani, Rebecchi (1993) with a new approach by joining molecular and indispensible traditional methods, light microscopy, and scanning electron microscopy. Differences in adult animals and, above all, egg shell morphology, and the peculiar cox1 sequence indicate that this population clearly belongs to a new species, M. vladimiri sp. n., which is here described. The results provide an example of how modern taxonomic and biogeographical research can be carried out on this animal phylum and in general on the animals belonging to the so-called meiofauna,in which morphological characters are often very few. This is the first tardigrade species to be described and barcoded contemporarily.

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 - Thermotolerance and hsp70 heat shock response in the cold-stenothermal chironomid Pseudodiamesa branickii (NE Italy) [Articolo su rivista]
Bernabò, P.; Rebecchi, Lorena; Martínez Guitarte, J. L.; Jousson, O.; Lencioni, V.

To better understand the physiological capability of cold-stenothermal organisms to survive high temperature stress, we analyzed the thermotolerance limits and the expression level of HSP70 gene under temperature stress of Pseudodiamesa branickii, an alpine stream’s species of Chironomids (Diptera).The effect of short-term high temperature shock on survival was examined in larvae of this species. The lethal temperature (LT100) and the lethal temperature 50% (LT50) for short-term exposure were 36.0 and 32.2°C, respectively. In addition, we tested the effects on survival of long-term shock and the hypothesis that a short pre-exposition to less severe high temperature would generate higher thermotolerance. The duration of the exposure influenced negatively the survival, whereas a prior exposure to a less severe high temperature generated an increase of survival, with a lethal temperature 2°C higher. To point out the molecular basis of this high thermal tolerance, the expression of the HSP70 gene family, including the constitutive (HSC70) and the inducible (HSP70) forms, was surveyed via quantitative RT-PCR after short term and long term exposure to high temperature. Larvae of P. branickii showed significant up-regulation of both members of the HSP70 family expression in response to increased temperatures, whereas in response to increased time of exposure a decrease of the expression level was underlined. Whereas previous studies have shown that cold-stenothermal Antarctic organisms lack the classical heat shock response, our results indicate that this cold-stenothermal alpine chironomid maintained an inducible heat shock response at a level potentially set during its temperate evolutionary past.

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 - A model study for tardigrade identification [Relazione in Atti di Convegno]
Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele

Using tardigrades from a single moss sample as a case study,we propose a new method for tardigrade species identification, which is often problematic, due to the low number of orphological characters. Identification at generic level was carried out on adults, while morphological analyses were performed on animals (LM) and eggs (LM and SEM), including hologenophores, vouchers used also for molecular analysis of COI mtDNA. This multi-approach method revealed the presence of three species of the“Macrobiotus hufelandi group” instead of the two species identified in a previous study. The validity of the method is shown, indicating that it could be applied to studies of problematic meiofauna taxa.

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 - 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, 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 - Tardigrada [Capitolo/Saggio]
D., Nelson; Guidetti, Roberto; Rebecchi, Lorena

.The chapter reports the following data about classification and ecology of Tardigrada of North America: Evolution, Major Morphological Taxonomic Characters (Claws, Buccal-Pharyngeal Apparatus, Cuticle,. Eggs), Anatomy, Digestive System, Other Anatomical Systems, Latent States (Cryptobiosis, Diapause), Reproduction and Development (Sexual Conditions, Reproductive Apparatus, Sexual Dimorphism, Gametogenesis and Gametes, Mating and Fertilization, Reproductive Modes, Eggs and Parental Care, Development, Post-Embryonic Development), Ecology ( Molting, Life History, Cyclomorphosis, Habitats, Population Dynamics, Trophic Relationships, Biogeography, Dispersal), Techniques, Collection, Extraction, Microscopy, Culturing, Molecular Analyses for Determining Tardigrade Phylogeny and Species Identification, Identification, Taxonomic Key to Genera of Freshwater and Terrestrial Tardigrada.

2009 - DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani &amp; Rebecchi 1993 (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Cesari, Michele; Bertolani, Roberto; Rebecchi, Lorena; Guidetti, Roberto

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani &amp; Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a very closely related species confirm the possibility of identifying a specimen of this species by its COI sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.

2009 - Expression of the 70 kDa Heat shock protein family in Alpine freshwater chironomids (Diptera, Chironomidae) under natural conditions [Articolo su rivista]
V., Lencioni; D., Boschini; Rebecchi, Lorena

Chironomidae represent up to 100% of the fauna of Alpine streams. Because they survive stress conditions such as extremely low temperature (annual mean &lt; 4°C), these organisms represent a good organism model to analyze the relationship between adaptations to cold and expression of stress proteins such as the 70 kDa Heat shock protein family. Fourth instar larvae of ten species of cold-stenothermal chironomids (Pseudodiamesa branickii, Diamesa latitarsis, Diamesa laticauda, Diamesa cinerella, Diamesa insignipes, Diamesa zernyi, Diamesa vaillanti, Orthocladius (Orthocladius) frigidus, Orthocladius (Euorthocladius) thienemanni and Paratriclocladius nivalis) were collected in a glacier-fed stream in N.E. Italy at two stations (1300 and 2600 m a.s.l.) and in two seasons (summer 2005 and spring 2006). Immunodetection and quantification of therelative levels of Hsp70 family were performed via Western blotting. Significantly different levels of Hsp70 were detected among species. The highest amounts were recorded in P. nivalis and D. insignipes, the lowest in P. branickii. Within the genus Diamesa, lower levels of Hsp70 were observed in the most cold stenothermal species than in the less cold stenothermal ones. These differences are explained by different autoecology. The results provide information on biochemical strategies of alpine midges to face cold temperatures under natural conditions and new insights into their possible response to global warming.

2009 - Stress response of a boreo-alpine species of tardigrade, Borealibius zetlandicus (Eutardigrada, Hypsibiidae) [Articolo su rivista]
Rebecchi, Lorena; D., Boschini; Cesari, Michele; V., Lencioni; Bertolani, Roberto; Guidetti, Roberto

Invertebrates living in extreme environments as well as those living under unpredictable habitat conditions must be able to survive severe environmental stresses bound to their habitats. Tardigrades represent a good animal model to analyze responses evolved by organisms to overcome extreme environmental stresses or to colonize extreme environments because they respond to desiccation or freezing in their habitats by entering cryptobiosis. The responses to environmental stresses have been evaluated almost exclusively in terrestrial tardigrades, while very little is known about the ability of limnic species to tolerate those stresses. This study evaluates the responses of the limnic boreo-alpine species Borealibius zetlandicus, under lab conditions, to stresses imposed by desiccation and temperature variation (freezing and heating). Our results indicate that active specimens are able to freeze, confirming the cryobiotic ability of this species. There is a negative correlation between survival and cooling rates. In contrast, no specimens of B. zetlandicus are able to survive desiccation. With regard to thermal tolerance, the animals show a high ability to resist heat-shock (LT50 = 33.0  0.5°C) for a short time. This wide tolerance to different environmental parameters could be the reason for the wide distribution of the species. Due to the disjunct distribution of the species and to the presence of cryptic tardigrade species that could have different ecological and physiological responses, we decided to characterize the population studied from a molecular point of view by investigating its COI mtDNA sequences.

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 - 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 - Cryptic Species in Macrobiotus (Tardigrada): A Morphological and Molecular Approach [Abstract in Atti di Convegno]
Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele

In the last years, morphological analysis of Tardigrada has been carried out even more in depth, leading to recognise many new species, thus reconsidering the concept of intraspecific variability. In addition, molecular approach has very recently identified a more complex scenario within some taxa, supposing or evidencing the presence of cryptic species. Our previous studies already identified a complex situation bound to karyology and reproductive biology within single morphospecies. For these reasons we have reconsidered populations belonging to the morphospecies Macrobiotus richtersi and to Macrobiotus “hufelandi group” analysing their morphology and mtDNA COI (cytochrome oxidase I) gene sequences. The presence of both diploid amphimictic and triploid apomictic populations has been confirmed in both taxa. In M. richtersi all animals are very similar, while limited differences have been evidenced in the egg shell ornamentations of some populations. DNA sequences have shown that populations with different reproductive modes (and ploidy degree) have always decidedly different haplotypes. Within parthenogens equal or very similar haplotypes are retrieved, while within amphimictics both equal and different haplotypes are found. In M. “hufelandi group” molecular analysis confirms the morphological diagnosis of 3 populations of Macrobiotus macrocalix, while it evidences differences between 2 populations attributed in references to Macrobiotus terminalis and M. cf. terminalis. A general conclusion is that in tardigrades it is important to integrate morphological and molecular analyses. In fact, in some instances the morphological approach is adequate to recognize the species, but sometimes the molecular approach is indispensable to identify cryptic species.

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 - Primo esempio di DNA barcoding nei tardigradi: un approccio morfologico e molecolare. [Abstract in Atti di Convegno]
Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele

La tassonomia dei tardigradi è attualmente basata sull’analisi di caratteri morfologici. In questi ultimi anni lo studio di questi caratteri è stato notevolmente approfondito, portando ad un notevole incremento delle specie descritte. Ciononostante, l’analisi morfologica non sembra in grado di risolvere tutti i problemi di identificazione delle specie. Già erano note morfospecie comprendenti popolazioni con diverso grado di ploidia e/o diverse modalità riproduttive. Recentemente, mediante indagini molecolari, è stata riconosciuta la presenza di specie criptiche. Messa a punto la tecnica di estrazione di DNA da singoli animali e da singole uova, abbiamo pertanto ritenuto opportuno iniziare uno studio di DNA barcoding, considerando esemplari di due specie raccolti nel locus typicus ed in altre località. Le due specie considerate sono state Macrobiotus macrocalix (locus typicus Gaianello, Modena) e Macrobiotus richtersi (locus typicus Clare Island, Irlanda). Sulle popolazioni di queste due e di altre località sono state condotte indagini morfologiche e molecolari (mtDNA COI). Entrambe le analisi hanno confermato l’attribuzione a M. macrocalix di quattro popolazioni, tre italiane ed una svedese. In M. richtersi, differenze sono state invece rilevate a livello molecolare tra la popolazione di Clare Island e le diverse popolazioni italiane, risultate differenti anche tra loro.

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.

2008 - Variation in anhydrobiotic survival of two eutardigrade morphospecies: a story of cryptic species and their dispersal [Articolo su rivista]
Faurby, S; K. I., Joensson; Rebecchi, Lorena; P., Funch

Studies of geographic variation in anhydrobiosis may increase our understanding of the population dynamics of terrestrial meiofauna and the relative importance of local adaptation and microhabitat niche separation. Alltough anhydrobiosis in tardigrades has been studied intensively, few studies have dealt with intraspecific variation in survival and none of these included genetic data, which are necessary when working with meiofauna as cryptic species are common. We analyzed the anhydrobiotic survival and genetic variation in COI of two eutardigrades (Richtersius coronifer and Ramazzottius oberhaeuseri) from Italy and Sweden to detect possible local adaptation. Survival was analyzed as a multidimensional contingency table and showed that anhydrobiotic survival was higher in Sweden for R. oberhaeuseri whereas no geographic variation was found for R. coronifer. Our genetic analysis indicated the coexistence of two cryptic species of R. oberhaeuseri in Italy, of which only one was found in Sweden. It could not be determined if the variation in Ramazzottius is intra- or interspecific due to the presence of these cryptic species. We suggest that geographic variation in anhydrobiotic survival may be a general phenomenon in tardigrades but further research is necessary to determine the degree of intraspecific variation. Our analysis showed indications of long term isolation of the individual populations of R. coronifer but recent dispersal in one of the cryptic species of Ramazzottius. We found higher survival in R. oberhaeuseri than in R. coronifer and suggest that these results can be explained by a coupling between anhydrobiotic survival and dispersal rate.

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 - Introduction to the Proceedings [Prefazione o Postfazione]
G., Pilato; Rebecchi, Lorena

2007 - Proceedings of the Tenth International Symposium on Tardigrada [Curatela]
G., Pilato; Rebecchi, Lorena

The Tenth International Symposium on Tardigrada took place in Catania, Italy from 18 to 23 June 2006. There were seventy-one participants at the Symposium, representing fourteen countries. The Symposium provided an unique opportunity both to review the progress in the tardigrade studies and to allow all delegates to meet and to discuss topics of common interest. The symposium included thirty-four lectures and thirty-one poster presentations on a wide variety of topics on tardigrade biology. Topics included systematics, morphological and molecular phylogeny, faunal studies, biogeography, ecology, survival strategies, and modern molecular methodologies in tardigrade research. The twenty-four papers appearing in the this special issue of the Journal of Limnology represent most of the contributors present in Catania. The Symposium offered an interesting Round Table on the systematic criteria of Tardigrada, and the opportunity to examine and to compare type materials from several tardigrade collections: Pilato and Binda’s collection, Bertolani’s collection and, thanks to the Museo Civico di Storia Naturale di Verona, Ramazzotti’s collection, Maucci’s collection and Biserov’s collection. Moreover, many tardigradologists have contributed bringing their material.

2007 - Shock da calore e stenotermia fredda: Pseudodiamesa branickii (Diptera, Chironomidae), un caso studio [Abstract in Atti di Convegno]
P., Bernabò; J. L., Martinez Guitarte; Rebecchi, Lorena; V., Lencioni

L’ambiente alpino può essere considerato un laboratorio particolarmente sensibile per l’osservazione e lo studio degli effetti dei cambiamenti climatici. Ci si attende infatti che le specie stenoecie che li colonizzano mostrino delle risposte ai cambiamenti climatici in atto più rapide rispetto a specie a maggiore valenza ecologica. L’aumento della temperatura dell’aria e la modificazione del regime delle precipitazioni, a cui stiamo assistendo a livello globale negli ultimi decenni, stanno provocando il ritiro dei ghiacciai e l’alterazione del regime idrologico e termico dei corsi d’acqua. In particolare, si prevede che i corsi d’acqua alimentati da fusione glaciale, attualmente interessati da un aumento delle portate e una diminuzione della temperatura dell’acqua, a medio-lungo termine mostreranno minori portate e temperature più alte a cui le specie che li popolano saranno esposte. Scopo di questo lavoro è stato quello di studiare la risposta a shock termico da calore in termini di sopravvivenza ed espressione genica (hsp70) in larve di una specie stenoterma fredda di Chironomide, Pseudodiamesa branickii, raccolto in torrenti alpini glaciali. Si è osservato che la sopravvivenza di larve al III-IV stadio esposte a shock termico diretto (1 ora) diminuisce all’aumentare della temperatura di stress (100% di sopravvivenza a 26°C e 0% a 36°C, temperatura letale) con un valore di LT50 pari a 32,2 ± 1,6 °C. Una sopravvivenza inferiore è stata registrata per larve al I e II stadio della stessa specie. L’ipotesi che una precedente esposizione a temperature intermedie tra quella di allevamento e quella di shock potesse generare un aumento della sopravvivenza alle alte temperature è stata testata esponendo le larve a 15 °C per 1 ora prima del trattamento di shock, osservando una temperatura letale 2°C superiore a quella ottenuta per gli shock diretti. Sono state inoltre condotte delle prove di resistenza a shock prolungati che hanno evidenziato una mortalità pari al 100% dopo 32 ore a 26°C e dopo 8 ore a 32°C.Tramite la tecnica della RT-PCR è stata infine analizzata l’espressione genica della famiglia di proteine Hsp70 (Heat shock protein 70kDa) evidenziando profili di espressione diversi per la forma inducibile (hsp70) e per la forma costitutiva (hsc70). La forma inducibile, come atteso, ha mostrato un andamento crescente all’aumentare della temperatura di shock, mentre la forma costitutiva, maggiormente espressa in larve non sottoposte a stress, ha mostrato un’iniziale induzione e una successiva decrescita all’aumentare della temperatura.Informazioni di questo genere possono essere funzionali alla realizzazione di modelli previsionali sulla perdita di biodiversità futura associata ai cambiamenti climatici global

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.

2007 - Ultrastructure of the digestive system of Ramazzottius tribulosus and Macrobiotus richtersi (Eutardigrada) in relationship with diet [Articolo su rivista]
A. M., Avdonina; N. M., Biserova; Bertolani, Roberto; Rebecchi, Lorena

The ultrastructure of the digestive system of tardigrades was already described in some species, but it has never been studied in relationship to the diet. Therefore, utrastructural analyses of the midgut and hindgut of the phytophagous Ramazzottius tribulosus and the zoophagous Macrobiotus richtersi have been made. In addition, the foregut of R. tribulosus has been also analyzed. For this part new ultrastructural details have been evidenced. Among them, distinct transverse pillar-like structures, lacking in the electron-dense and compact cuticle of the buccal tube; a hole or groups of holes sometime present in the buccal tube; a large cavity within each salivary glands, where secreted mucus is accumulated; one valve made up by folds of the pharynx and located at the transition from pharynx to esophagus, already found in the zoophagous Isohypsibius prosostomus. In both analyzed species the increase of midgut surface is realized by two orders of folds of the gut wall and by microvilli. In R. tribulosus there are many first-order folds and few second-order fold, whereas in M. richtersi we found an opposite pattern. A perithrophic membrane and microvilli with a well developed glycocalix are found only in the midgut lumen of R. tribulosus. The density of microvilli and the ratio between the real surface with microvilli and the hypothetic surface without microvilli is lower in the zoophagous M. richtersi and I. prosostomus than in the phytophagous R. tribulosus. All these data represent an indirect indication of differences in digestion physiology between phytophagous and zoophagous tardigrade species. The shape of the hindgut is similar in both species and in particular the lumen of the hindgut looks as a heart-like cavity with some narrow cell evaginations.

2006 - Cryobiosis survival in tardigrades. [Abstract in Atti di Convegno]
Bertolani, Roberto; Rebecchi, Lorena; Boschini, Deborah; Guidetti, Roberto

Eight species of eutardigrades collected in different terrestrial and freshwater habitats and characterized by different anhydrobiotic capabilities have been submitted to freezing and thawing experiments. The aim was to test: i. the interspecific capability to survive cryobiosis and the differences in recovery time to active life; ii. the relationship between survival and cooling rates. Starting from a uniform condition of tardigrades maintained for 24 h in water at 14°C, the cooling was performed according to three different protocols. Experiment 1: replicates for each species considered were put in 4 ml of water, frozen at three temperatures (-9°C, -20°C and -80°C) and kept frozen for six days. Experiment 2: replicates of Ramazzottius oberhaeuseri and Amphibolus volubilis were put in 4 ml of water, frozen in liquid nitrogen for two minutes and then stored at –9°C. Experiment 3: replicates of R. oberhaeuseri were placed in different amounts of water (2 ml, 1 ml and 0.5 ml), frozen at three different temperatures as in experiment 1 and kept frozen for six days. Before thawing, all frozen animals were firstly put or maintained at –9°C and then thawed at 14°C. Animals were examined both after 2.5 h and 24 h, considering alive the animals with evident and coordinated body movements. In experiment 1, R. oberhaeuseri, A. volubilis, Macrobiotus areolatus and Macrobiotus richtersi show a high survival at any tested temperature. Hypsibius dujardini, Borealibius zetlandicus and Diphascon cf. scoticum show a lower survival and differences in survival among each temperature. Survival of Dactylobiotus parthenogeneticus is null at any tested temperature. In most cases, the recovery time increases with the fall of the temperature and differs among the species. The differences among the species seem clearly related to the habitat and above all to the capability to carry out anhydrobiosis, suggesting a strong relationship between anhydrobiosis and cryobiosis. In experiment 2, all specimens of both species did not survive to freezing at –196°C. In experiment 3, R. oberhaeuseri shows high survival also when the animals are frozen in less than 4 ml of water, even though a survival decrease is recorded between –9°C and –80°C for all tested water amounts. In general, the survival is lower when the water freezing time is shorter (corresponding to a higher cooling rate). Moreover, a negative relationship between the water freezing time and the recovery time has been found: the shorter the water freezing time, the longer the recovery time. A too short water freezing time does not allow survival. These results can be easily explained by the need to produce a sufficient amount of protectants, or not enduring too many damages.

2006 - Cysts of Amphibolus volubilis (Eutardigrada, Eohypsibiidae): seasonal dynamics and laboratory induction. [Abstract in Atti di Convegno]
Guidetti, Roberto; Boschini, Deborah; Bertolani, Roberto; Rebecchi, Lorena

It is known that specimens of Amphibolus can carry out both cryptobiosis (anhydrobiosis and cryobiosis) and encystment and that they produce two types of cysts (type 1 and type 2). In Amphibolus nebulosus from Greenland, the type 1 cyst is related to winter and the type 2 to summer. In Amphibolus volubilis, found in Apennines (Monte Rondinaio, Italy), a detailed description of the encystment processes has been done, but not of the relationships between climatic factors and dynamics of the cyst 1 and cyst 2. Therefore, a study on seasonal dynamics of the A. volubilis cysts has been carried out with monthly samplings, from March 2003 up to March 2005. For each sampling, all specimens present in five-six replicates of 0.5 g of moss have been collected and analyzed.In all samplings, non-encysted and encysted animals (type 1 or type 2 cysts) have been found. The cyst trends are similar in the two considered years. Type 1 cysts have been found from November to March-April, but they were present in a very low percentage with respect to the total animals. Type 2 cysts have been found from June to October and are often more abundant than the non-encysted tardigrades. The two types of cysts never overlap. Correlation tests have evidenced that dynamics of type 2 cyst is positively related to the air temperature (T) and negatively related to the relative humidity (RH) of the air. The trend of type 1 cyst is negatively related to T, while no relationship to RH has been evidenced. The presence of non-encysted animals is negatively related to T and positively related to RH. Experiments of encystment induction have been performed in laboratory. In the same experimental conditions (same temperature, photoperiod, food and oxygen availability), non-encysted animals collected in April form type 2 cysts, whereas animals collected in November form type 1 cysts. The number of days to enter each type of cyst is related to experimental temperature. At the same temperature, the animals spent less time to enter in type 1 cyst than in type 2 cyst. In lab, the animals come out from type 2 cyst, if do not die, soon or later encyst again and enter always type 1 cyst.The data from nature and those from laboratory lead to conclude that, differently from what happens in anhydrobiosis and cryobiosis, climatic conditions do not directly affect encystment; but seasonal changing induces still unknown endogenous factors responsible of that kind of dormant stage.

Boschini, Deborah; Guidetti, Roberto; Rebecchi, Lorena

Invertebrates living in extreme habitats at high altitudes or latitudes, as those living in ephemeral habitats, must be able to survive environmental stresses such as desiccation and freezing. In this contest several important scientific issues are emerging. Organisms can have four responses to overcome environmental stresses: regulative, acclimation, developmental, and evolutionary responses. Regulative and acclimation responses are carried out by the organisms in short or very short time to maintain their internal conditions for appropriate optimal functioning. Both responses are reversible in such a way as to follow the environmental fluctuations. They are also measurable according to a space-time scale depending on the size and the length of the life cycles of the organisms. Considering tardigrades, their persistence in unpredictable habitats is due to two widespread adaptive strategies, which are regulative responses: ability to enter cryptobiosis (both anhydrobiosis and cryobiosis) and/or to enter cyst. Anhydrobiosis and encystment are certainly characterized by several molecular events only partly identified. Other than the disaccharide trehalose, several stress proteins seem to be further keys to understand anhydrobiotic mechanisms. In particular, heat shock proteins (Hsps) and their molecular partners, which play diverse roles, including that of molecular chaperons, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion and degradation of other proteins.A moss-dwelling eutardigrade collected in Northern Apennines (Modena, Italy; 1700 m a.s.l.), Amphibolus volubilis, has been utilized in our lab to evaluate the survival strategies in unpredictable habitats. It is known that this species is able to enter anhydrobiosis and cryobiosis and to form cysts. Lab experiments on desiccation tolerance have been carried out considering three different values of air relative humidity (RH). Survival resulted directly related to the RH values, and high survival (about 80%) was obtained only with the highest RH value tested (85%). Desiccated animals were utilized to evaluate the Hsp70 and Hsp90 expression by means of SDS-Page and Western blotting analysis. Hsp expression was also evaluated in active animals and (only Hsp70) in encysted (type 2 cyst) animals. Quantitative comparisons of protein expression have been made among these three conditions. The level of both Hsp70 and Hsp90 is higher in desiccated animals than in active ones. Encysted animals have a Hsp70 level lower than both active and desiccated animals. These results indicates that Hsp induction due to desiccation is necessary to preserve cells from desiccation damages. Instead, the lower Hsp level found in cysts with respect to active animals could be explained hypothesizing a little need of these proteins in animals already protected from environmental stresses by strong cuticular layers.

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 - Encystment processes and the Matrioshka-like stage in a moss-dwelling and in a limnic species of eutardigrades (Tardigrada) [Articolo su rivista]
Guidetti, Roberto; D., Boschini; Rebecchi, Lorena; Bertolani, Roberto

Tardigrades have two forms of dormancy, namely cryptobiosis and encystment. The encystment is a form of diapause known for a limited number of species of tardigrades and still little studied. To increase the knowledge on encystment, two species of eutardigrades from Italy have been considered: the moss-dwelling Amphibolus volubilis (Eohypsibiidae), and the limnic Dactylobiotus parthenogeneticus (Murrayidae). Cysts have been collected in nature, or induced under laboratory conditions. In the latter case, it was possible to follow the several steps of encystment processes. Two different types of cyst (type 1 and type 2) have been found in A. volubilis, while in D. parthenogeneticus only one type has been found. In general, the ovoid-shaped cysts are constituted by a series of cuticles surrounding the animals and resemble an onion or a Matrioshka Russian doll. In all three types of cyst, the encystment processes show both common and peculiar traits. Encystment begins with the discharging of the sclerified parts of the buccal-pharyngeal apparatus, as in the molting process, but without the loss of the old cuticle. Then, two or three new cuticles are serially synthesized, according to cyst type. In A. volubilis, the ultrastructure of these new cuticular involucra is similar to that of non-encysted animal cuticles, while in D. parthenogeneticus the ultrastructure of the new cuticular involucra differs from that of non-encysted animal cuticle. A modified buccal-pharyngeal apparatus has been observed both in type 2 cyst of A. volubilis and in the D. parthenogeneticus cyst.

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 - Geonemy, ecology, reproductive biology and morphology of the tardigrade Hypsibius zetlandicus (Eutardigrada: Hypsibiidae) with erection of Borealibius gen. n. [Articolo su rivista]
G., Pilato; Guidetti, Roberto; Rebecchi, Lorena; O., Lisi; Jg, Hansen; Bertolani, Roberto

The morphology, biology and geographic distribution of Hypsibius zetlandicus (Murray, 1907) are considered. LM and SEM analyses have been carried out on H. zetlandicus and other two species of Hypsibius, with particular emphasis on the buccal-pharyngeal apparatuses. Some peculiar characteristics of this apparatus in H. zetlandicus lead us to the erection of the new genus Borealibius, to which H. zetlandicus (Borealibius zetlandicus comb. n.) is transferred.In the light of new findings in polar, sub-polar and alpine regions and the available bibliographic references, we evidenced for this species a boreo-alpine distribution. The presence of uncommon phenomena for a tardigrade species have been observed analyzing traits of B. zetlandicus reproductive biology (presence of hermaphroditism and parental cares) and ecology (colonization of very different substrates).

A., Avdonina; N. M., Biserova; Rebecchi, Lorena

The morphology of the bucco-pharyngeal apparatus is correlated with the food utilized. Nevertheless, even though the ultrastructure of the digestive system of tardigrades was already described in some species, it was not studied in depth, and not in relationship to the feeding. Therefore, comparative utrastructural analyses of the digestive system of the phytophagous Ramazzottius tribulosus and the zoophagous Macrobiotus richtersi were made.In R. tribulosus within the cuticle of the buccal ring there are distinct transverse pillar-like structures. Posterior to the mouth, the cuticle of the buccal tube becomes electron-dense and compact, without pillar-like structures and vacuoles, even though holes or groups of holes are sometime present anterior to the stylet supports. At each side of the buccal tube there are thin processes of the salivary glands, which form a large reservoir where secreted mucous is accumulated, like in the salivary sac of arthropods. Under the reservoir wall there are thin muscle processes and neuromuscular contacts. Stylets and stylet supports are located in the salivary reservoirs. Each stylet has a basal cuticular part and an apical hard calcium part divided by a well visible border. The beginning of the pharynx is characterized by thin valves and cuticular apophyses; the latter are homogenous ultrastructure so as the posterior part of the buccal tube and placoids. The lumen of the pharynx is limited by a clearly tri-layered thin cuticle. At the transition from pharynx to oesophagus there is one valve formed by folds of the gut which hinder the food return. The increase of midgut surface is realized by both large folds of the gut wall and microvilli. Microvilli are relatively short and thick but have well developed glycocalyx and in cross section look disposed regularly forming very compact hexagons (111 per μm2). Epithelial cells of the midgut have large and small vacuoles. In tardigrades fixed after feeding, the midgut lumen exhibits a perithrophic membrane. In the cross section the lumen of the rectum looks as a big heart-like cavity with some narrow cell evaginations. The lumen is lined by a thin cuticle.For M. richtersi, at this moment we have considered only the ultrastructure of midgut and hindgut. Microvilli of the midgut are shorter and thinner than those of R. tribulosus. They form less dense hexagons (73 per μm2). Glycocalyx is present but not particularly developed. The ultrastructure of the midgut (epithelial cells, peritrophic membrane) and that of the rectum, is similar to what is found in R. tribulosus. Comparing these results among them and with published data on the zoophagous Isohypsibius prosostomus, several similarities has been found, such as valves, cuticle lining, ultrastructure of epithelial cells, and peritrophic membrane. They can be related to common feeding processes. The presence of peritrophic membrane indicates that in tardigrades extracellular digestion occurs. Nevertheless, we can identify differences that can be related to the food utilized. Mainly, there is more absorption surface of the midgut in the phytophagous R. tribulosus than in zoophagous M. richtersi and I. prosostomus. Investigations supported by INTAS Grant № 04-83-3807 to A.A.

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

A., Avdonina; N. M., Biserova; Rebecchi, Lorena

The current importance of investigating the anatomical and ultrastructural organization of Tardigrada nervous system is determined in relationship to the early evolution of the arthropods. According to the data by Dewel and Dewel (1996, 1999), the organization of the Tardigrada brain could be best understood by analyzing the pattern of the nervous system in the trunk. On this basis, we analyzed the ultrastructure of the trunk ganglia in the eutardigrade Macrobiotus richtersi. The trunk ganglion is located ventrally under the midgut beneath the delicate visceral muscles and surrounded by the body-cavity. The ganglion has an oval compact shape and distinct basal lamina. Eight perikarya have been found in the investigated portion of the ganglion (25 serial sections were examined). Most of them are located at the periphery and they contact basal lamina directly. At least one of the neurons is situated to some extent deeper and it contacts fringe cells. This neuron projects the process to the center of the neuropile and then passes into the lateral nerve. Usually, perikarya have an oval nucleus and neuroplasm with dense packed organelles such as mitochondria, reticulum and vesicles. Only one cell has irregular-shaped nucleus and a lucent cytoplasm. It is unclear if the Tardigrada nervous system has glial cells or not. Perhaps this lucent cell has a glia-like function in the trunk ganglion of M. richtersi.A pair of nerves goes out from the ganglion and innervates the lateral area of the segment. These nerves have no neuron cell bodies and any other cells like glial cells. The nervous processes are slim, dense-packed and contain mitochondria and synaptic vesicles. A basal lamina surrounds not only the ganglion but nerves too, and consists of fine dense filaments. Rough fibres have not been found. There are numerous neurites containing small clear and rare dense vesicles in the neuropile. Besides in the ganglion there is one large axon which contains clear cytoplasm and dense-core vesicles. Its perikaryon is not found in the ganglion. This axon goes through the ganglion and passes out into a nerve. Many processes in the neuropile have a specific dense neuroplasm with clear small vesicles. We have found a similar pattern in neurites innervating the salivary duct and muscles of the salivary glands and perhaps in the circumesophageal connective of another eutardigrade, Ramazzottius tribulosus. Investigations partially supported by INTAS Grant № 04-83-3807 to A.A.

2006 - Tenth International Symposium on Tardigrada [Esposizione]
G., Pilato; Bertolani, Roberto; G., Binda; Guidetti, Roberto; O., Lisi; Rebecchi, Lorena

The Tenth International Symposium on Tardigrada took place in Catania, Italy from 18 to 23 June 2006 and was held in the Auditorium of Benedettini, a fascinating historic building in Catania’s downtown. There were seventy-one participants at the Symposium, representing fourteen countries. The Symposium provided a unique opportunity both to review the progress of tardigrade studies and to allow all delegates to meet and to discuss topics of common interest. The symposium included thirty-four lectures and thirty-one poster presentations on a wide variety of topics in tardigrade biology. Topics included systematics, morphological and molecular phylogeny, faunal studies, biogeography, ecology, survival strategies, and modern molecular methodologies in tardigrade research. The twenty-four papers appearing in this special issue of the Journal of Limnology represent most of the contributors present in Catania.

2006 - Tenth International Symposium on Tardigrada: Catania, Italy 18-23 June 2006 [Articolo su rivista]
Pilato, G.; Bertolani, R.; Binda, M. G.; Guidetti, R.; Lisi, O.; Rebecchi, L.

2006 - Thermal tolerance and expression of Hsp70 in larvae of Diamesa spp. [Abstract in Atti di Convegno]
V., Lencioni; Boschini, Deborah; B., Maiolini; Rebecchi, Lorena

Air temperature and precipitation patterns are likely to change under a scenario of global climate warming, with consequent effects on water temperature and hydrology of running waters, especially of those fed by glaciers. Invertebrates living in these habitats (mainly Chironomidae Diamesinae) are extremely specialised and have developed unique survival strategies to face environmental constraints. The ability to survive heat-shocks was investigated in cold stenothermal species, threatened of extinction by global warming. IV instar larvae of Diamesa cinerella gr. were collected seasonally (from March 2005 to March 2006) in the Noce river (Trentino, NE Italy) at two altitudes (1300 and 2600 m a.s.l.). Larvae were acclimated in a thermostatic chamber for 24 h at 4°C and then exposed for 1 h to heat-shocks from 26 to 35°C. Survival was recorded 1 h after the shock and thermal tolerance was evaluated as LT50. D. cinerella gr. resulted thermotolerant (LT50 varied from 30.1 to 32.9°C). Hsp70 were detected and quantified both in heat-shocked and un-shocked larvae of D. cinerella gr and in un-shocked larvae of other Diamesa species by means of SDS-Page and immunoblotting. An increase of Hsp70 expression was detected in shocked larvae of D. cinerella gr. collected in summer. Constitutive stress proteins were found in all species maintained at 4°C. This could explain the high resistance to brief heat-shocks found in D. cinerella gr.

2006 - Thermotolerance and thermal acclimation in active tardigrades. [Abstract in Atti di Convegno]
Boschini, Deborah; Bertolani, Roberto; Rebecchi, Lorena

The ability of desiccated (anhydrobiotic) tardigrades to survive and to resist high temperature stresses (up to 100°C) is well-known, while the tolerance of active (hydrated) tardigrades to heat-shocks is still very little known. In order to increase our knowledge on thermal adaptations of active tardigrades, we have carried out lab experiments on three eutardigrade species to evaluate the ability to survive to heat-shock (considering lethal temperature - LT50 - and critical thermal maximum - CTmax), and the possibility of thermotolerance induction. We considered two terrestrial species, the moss-dwelling Amphibolus volubilis and the lichen-dwelling Ramazzottius oberhaeuseri, and one limnic species, Borealibius zetlandicus. These species differ each other in anhydrobiotic and cryobiotic abilities, substrate colonized.Starting from a uniform condition of tardigrades maintained for 24 h in water at 16°C, groups of hydrated tardigrades have been exposed for 1 hour to a heat-shock (different experiments from 26°C to 42°C). The presence of active animals (body movements) was evaluated immediately after heat-shock (t0), after 1h (t1) and after 24 h (t24) from heat-shock. Survival was represented by active animals at t24. For A. volubilis and R. oberhaeuseri the presence of active animals and survival was evaluated also after acclimation of 1 h (the first species at 28°C, the latter at 30°C) and subsequent heat-shocking to temperatures higher than 33°C. All species look thermotolerant, even though their survival significantly decreases with the increase of the stress temperatures. Both CTmax and LT50 are species-specific. The first one is 39.0°C for A. volubilis and 37.0°C for R. oberhaeuseri and B. zetlandicus. The latter is 35.1°C for A. volubilis, 33.6°C for R. oberhaeuseri and 33.0° for B. zetlandicus. The percentage of active animals changes according to the shock temperature and differs among the species. The number of animals with active movements often significantly increases between t0 and t1 for some temperatures, apart R. oberhaeuseri which shows a decrease at 28°C. There are not significant differences between t1 and t24 in the number of active animals, with the exception of an increase in A. volubilis at 30°C and 33°C. Both in A. volubilis and R. oberhaeuseri the acclimation produces significant survival increases of 10-40% for heat-shock temperature between 33°C–39°C, while at 40°C and 42°C there is not survival. These results demonstrate that tardigrades, even when active, have evident ability to survive heath stresses, even though sampled in different habitats and characterized by quite different cryptobiotic performances.

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.

2005 - Ultrastructure of the spermatozoon of Talpa romana (Mammalia, Lipotyphla) [Articolo su rivista]
F., Beolchini; Rebecchi, Lorena; Bertolani, Roberto; E., Capanna

In this study, we used SEM and TEM to investigate the ultrastructure of spermatozoa from the cauda epididymis of Talpa romana. For comparison, we also analysed spermatozoa from the cauda epididymis of T. europaea captured in the same area. The male gamete of T. romana has a flattened head with an elliptic profile, consisting of a large acrosome and a nuclear region separated by a thin subacrosomal space. At the tip of the nucleus, the subacrosomal space ends in a finger-shaped projection. The tail includes a connecting piece, middle piece, principal piece and end piece. The male gametes of T. romana are substantially similar to those of T. europaea. A comparison with other species of insectivores permits extension of the similarity of sperm features to Scalopus aquaticus and Condylura cristata. Many spermatozoa from the cauda epididymis of T. romana and T. europaea have the tail bent at the annulus, and this is always associated with remnants of cytoplasmic droplets. This morphology is considered to be a common phenomenon.

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.

2004 - Tardigrades in extreme habitats: morphological and molecular aspects in anhydrobiotic and encysted Amphibolus volubilis. [Abstract in Atti di Convegno]
Rebecchi, Lorena; Boschini, Deborah; Guidetti, Roberto; F., Callegari; Bertolani, Roberto

In terrestrial ecosystems tardigrades often inhabit transient systems that can support life only for a fraction of the year (e.g. arctic tundra, deserts, temporary ponds, mosses, lichens and leaf litter). Persistence of tardigrades in these harsh habitats is due to their ability to enter dormant states such as cryptobiosis and/or encystment. Cryptobiosis is directly induced by exogenous stimuli (desiccation: anhydrobiosis; freezing: cryobiosis) and it is promptly broken when the adverse conditions are removed. Encystment is under endogenous control and could be only indirectly induced by environment stimuli. Breaking encystment requires a specific cue that may or may not correspond to favourable environmental conditions.Amphibolus volubilis is a moss-dwelling tardigrade with a boreo-alpine distribution. Therefore, it represents a good model species to study the survival strategies in unpredictable environments. We verified that it is able both to enter cryptobiosis and to form cysts. Morphological changes of the body have been evidenced in anhydrobiotic tuns and cysts collected in nature from moss collected in a post-glacial valley (Northern Apennines, Modena, Italy, 1700 m a.s.l.) and/or induced in lab. Entering anhydrobiosis specimens contract longitudinally turning their body into a tun, lose most of their free and bound water and reduce or suspend their metabolism. The cyst is the result of a series of successive and continued morphological changes that are more complex than those involved in tun formation. Encystment involves repeated de novo synthesis of new cuticular structures (several cuticles with different ultrastructure, modified and unmodified buccal-pharyngeal apparatuses and claws) and a reduction of metabolism and loss of water lower than those involved in the anhydrobiotic state. The expression of Heat shock proteins (Hsps) have also been investigated in A. volubilis. Hsp 70 has been detected in active, anhydrobiotic and encysted specimens, evidencing that the synthesis of Hsp 70 in this species is not only related to desiccation stress. The synthesis of Hsp 70 was evidenced only in the anhydrobiotic state of the tardigrade Richtersius coronifer and related to tun formation (Ramløv and Westh, 2001).

2004 - Twelfth International Meiofauna Conference [Esposizione]
F., Bertasi; Bertolani, Roberto; V. U., Ceccherelli; M., Colangelo; Guidetti, Roberto; Rebecchi, Lorena; Todaro, Mary Antonio Donatello

News insights on phylogenetic relationships and distribution patters of meiofaunal based on morphological and /or molecular analysis Coupling habitat complexity/heterogeneity and meiofaunal assemblagesMeiofauna in space and time Meiofauna trophic relationshipsDeep-sea meiofaunaResponse of meiofaunal taxa and/or assemblages to physical and/or chemical disturbanceMeiofauna in selected habitat

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.

2003 - The gametogenetic cycle of two syntopic populations of moles: Talpa romana and Talpa europaea (Mammalia, Insectivora, Talpidae) [Articolo su rivista]
F., Beolchini; Rebecchi, Lorena; Bertolani, Roberto; E., Capanna

The gametogenetic activity of two syntopic populations of moles, Talpa romana and T europaea (Mammalia, Insectivora, Talpidae), was compared. The testes and the ovaries of 42 specimens of T romana and of 41 specimens of T europaea were histologically characterised. Male gametogenetic activity was shown by the presence of spermatozoa in the epididymis. The diameter of seminiferous tubules was also measured. Female gametogenetic activity was evaluated on the basis of the number of secondary and pluristratified primary follicles in the ovary. Though not identical, the gametogenetic cycle of T romana largely overlaps that of T europaea. Moreover, for the two populations of moles a relationship between the duration of the reproductive activity and climatic and latitude-dependent parameters is suggested.

2003 - The spermatozoon of the Echiniscidae (Tardigrada, Heterotardigrada) and its phylogenetic significance [Articolo su rivista]
Rebecchi, Lorena; A., Guidi; Bertolani, Roberto

The spermatozoon ultrastructure of four species of moss-dwelling Heterotardigrada belonging to four genera of Echiniscidae, namely Pseudechiniscus juanitae, Echiniscus duboisi, Novechiniscus armadilloides and Antechiniscus parvisentus, was investigated. In all species, the testicular male gamete is similar in morphology and in length. The spermatozoon is made up of along head, consisting of a cylindrical acrosome and an oval or rod-shaped nuclear region which contains a nucleus with osmiophilic and electron-dense chromatin, and a tapering tail, with a 9+2 axoneme. An elongated sack-like structure originates from the posterior part of the head, extending beyond the main axis of the cell and running parallel to the tail. It consists of two parallel tubular regions which sometimes form a strict double helix and contain two voluminous, free mitochondria with unmodified cristae. In addition, a voluminous vesicle is present laterally to the centriole or between the end of the nucleus and the beginning of the mitochondria, limited by two cytomembranes and filled with electron-lucent and granular material. The male gametes representative of these mossdwelling Echiniscidae are very similar to the spermatozoa of the marine Echiniscoididae Echiniscoides sigismundi. This close similarity emphasises that habitat changes have had little influence on the organisation of the sperm cell representative of Echiniscoidea. Spermatozoon characters which could be useful for phylogenetic studies on Tardigrada are discussed.

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.

2002 - Experimentally induced anhydrobiosis in the tardigrade Richtersius coronifer: Phenotypic factors affecting survival [Articolo su rivista]
Ki, Jonsson; Rebecchi, Lorena

The ability of some animal taxa (e.g., nematodes, rotifers, and tardigrades) to enter an ametabolic (cryptobiotic) state is well known. Nevertheless, the phenotypic factors affecting successful anhydrobiosis have rarely been investigated. We report a laboratory study on the effects of body size, reproductive condition, and energetic condition on anhydrobiotic survival in a population of the eutardigrade Richtersius coronifer. Body size and energetic condition interacted in affecting the probability of survival, while reproductive condition had no effect. Large tardigrades had a lower probability of survival than medium-sized tardigrades and showed a positive response in survival to energetic condition. This suggests that energy constrained the possibility for large tardigrades to enter and to leave anhydrobiosis. As a possible alternative explanation for low survival in the largest specimens we discuss the expression of senescence. In line with the view that processes related to ahhydrobiosis are connected with energetic costs we documented a decrease in the size of storage cells over a period of anhydrobiosis, showing for the first time that energy is consumed in the process of anhydrobiosis in tardigrades.

2001 - Anhydrobiotic survival in populations of the tardigrades Richtersius coronifer and Ramazzottius oberhaeuseri from Italy and Sweden [Articolo su rivista]
Ki, Jonsson; S., Borsari; Rebecchi, Lorena

We report a study on anhydrobiotic survival in the eutardigrades Richtersius coronifer and Ramazzottius oberhaeuseri. In each of these species, we investigated the anhydrobiotic survival of two populations, one from Sweden and one from Italy. We found that anhydrobiotic survival was similar in the Swedish and the Italian populations in both species, indicating no divergence with respect to anhydrobiotic capacity. Body size had a strong effect on the probability to recover from anhydrobiosis, but the effect was in opposite direction in the two tardigrade species. Ramazzottius oberhaeuseri had a considerable higher overall survival (66%) than R. coronifer (40%). This result is in line with earlier studies that found R. oberhaeuseri to have a very high water-retentive capacity.

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.

2001 - Tardigrades as a model for experiences of animal survival in the space. [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena; Joensson, Ki; Borsari, S; Guidetti, Roberto

In this paper the biological characteristics of tardigrades are discussed in term of their ability to tolerate the stresses typical of the environmental space.

2001 - The spermatozoon in tardigrades: Evolution and relationships with the environment [Articolo su rivista]
Rebecchi, Lorena

The spermatozoon ultrastructure of seven species of tardigrades, one heterotardigrade echiniscid and six eutardigrades, was analysed and described. Tardigrade species were collected from freshwater sediments, moss and leaf litter. A phylogenetic evaluation was made and relationships of spermatozoon shape and habitat were discussed. The spermatozoon morphology of the moss-dwelling echiniscid (heterotardigrade) was very similar to that found in marine species of the same order. This indicates that in Echiniscoididae change of habitat had little influence on sperm structural organisation. In contrast, eutardigrade spermatozoa. have developed several different structural organisations that may be related to family and/or habitat.

2000 - Cuticle structure and systematics of the Macrobiotidae (Tardigrada, Eutardigrada) [Articolo su rivista]
Guidetti, Roberto; Rebecchi, Lorena; Bertolani, Roberto

The cuticle of tardigrades is characterized by three main layers: epicuticle, intracuticle and procuticle. Pillars are present in the epicuticle of almost all heterotardigrades, but these structures are also known in a few species of eutardigrades. The apparent heterogeneity of the cuticular ultrastructure in several species of the Macrobiotidae (Eutardigrada) prompted us to analyse the structure of the cuticle in this family. Eleven species in several genera were investigated with light and/or electron microscopy. All the species of the genera Murrayon and Dactylobiotus showed pillars in the epicuticle, whereas the examined species of Macrobiotus, Richtersius and Xerobiotus completely lacked pillars. Therefore, in the Macrobiotidae, in contrast to what appears with light microscopy, the cuticle is homogeneous within each genus examined at the electron microscopic level. Considering the absence of pillars in the Macrobiotidae a synapomorphy, we propose the erection of two new subfamilies. Macrobiotinae subfam. n. is characterized by the absence of pillars in the epicuticular layer and includes, in addition to the genera Macrobiotus, Xerobiotus and Richtersius, the genera Pseudohexapodibius, Adorybiotus, and probably also Minibiotus, Calcarobiotus and Pseudodiphascon. Murrayinae subfam. n. is characterized by the presence of pillars in the epicuticular layer and includes the genera n Murrayon, Dactylobiotus and, probably, Macroversum.

2000 - Effects of a sulfonylurea herbicide on soil microarthropods [Articolo su rivista]
Rebecchi, Lorena; Sabatini, Maria Agnese; C., Cappi; Grazioso, Pasqualina; A., Vicari; G., Dinelli; Bertolani, Roberto

The short-term effects on soil microarthropods of the herbicide triasulfuron, belonging to the chemical class of sulfonylureas, were evaluated in two fields which had never been treated with sulfonylureas, and were cultivated with winter wheat. In particular, the effects of single applications at rates corresponding to two- (rate 2) and sixfold (rare 6) the recommended agricultural rare (7.5 g active ingredient ha(-1)) were analysed and compared with controls. The changes in the populations of the main groups of microarthropods were evaluated. Rate 2 had very low effects, whereas rate 6 produced a significant decrease in the number of microarthropods, Acarina and Collembola in the surface soil layer (0-7.5 cm). The Collembola were analysed at the species level. Statistical analysis revealed significant differences only for a few species, and only after treatment with the highest rate of triasulfuron. Finally, the results of the field tests were compared to those of laboratory tests carried out previously, which examined the effects of the same herbicide on a collembola species.

2000 - Female gonad of moles, genus Talpa (Insectivora, Mammalia): Ovary or ovotestis? [Articolo su rivista]
F., Beolchini; Rebecchi, Lorena; E., Capanna; Bertolani, Roberto

The female gonads of moles (genus Talpa) are composed of a cortex, functioning as an ovary, and a medulla, which is structurally similar to that of the testis. In the female reproductive apparatus there are masculine glandular annexes, such as a bilobate prostate, two Cowper glands, and a penis-like clitoris. All these features have recently led to the hypothesis of the presence of hermaphroditism due to sex-reversal in Talpa. The purpose of this study is to understand the functional significance of the structural organization of the female gonads in order to verify this hypothesis. Histological, histochemical and ultrastructural analyses have been carried out on several gonads of both sexes of two species: T. europaea and T. romana, including three fetuses. In both species, the cortical region of the female gonad shows a regular oogenetic activity. While the medulla is composed of interstitial cells that are partly organized in cord-like structures, no spermatogenetic activity has been ever observed inside of them. A histochemical analysis shows that in both sexes the interstitial cells secrete steroids, presumably estrogens as well as androgens. The presence of androgens in the female gonads would therefore explain the persistence of male glandular annexes in the female reproductive apparatus and both the sexual and behavioral monomorphism typical of the genus Talpa. Nonetheless, the female gonad of moles is a real ovary and a well-defined gonochorism exists. Therefore, there is no reason to assert the presence of hermaphroditism due to sex reversal. J. Exp. Zool. 286:745-754, 2000.

2000 - Maturative pattern of the ovotestis in two hermaphrodite species of eutardigrades [Articolo su rivista]
Rebecchi, Lorena; Guidi, A; Bertolani, Roberto

We studied the maturative pattern of the ovotestis and the reproductive cycle of two hermaphrodite species of eutardigrades belonging to different families and collected independently from a chestnut litter. A long-term study on Macrobiotus joannae (Macrobiotidae), collected monthly, and a short-term study on Amphibolus weglarskae (Eohypsibiidae) were carried out. In both species the reproductive apparatus is represented by an ovotestis and by a short gonoduct. A caudal sack-like evagination of the ovotestis was found only in M. joannae. The size of the gonad varied in relation to the body size and degree of maturation of the germinal elements. In both species the maturative pattern of the ovotestis can be subdivided into four stages. The first stage includes three phases (undifferentiated, male and previtellogenic phases), whereas stages 2-4 correspond to the stages of vitellogenesis. Groups of 3-8 oocytes mature in strict relation to the molting cycle. In both species there are at least two cycles of egg production, indicating that hermaphroditic tardigrades are iteroparous. Apart from the first two phases of stage 1, all stages simultaneously possess male and female germinal elements which are not separated by barriers. The gonad is exclusively male only in relatively small specimens of both species, indicating that the male phase (m) is present only in the first reproductive cycle. The contemporary presence of mature oocytes and spermatozoa in close contact with each other is evidence that tardigrades are simultaneous hermaphrodites, and it suggests the existence of self-fertilization. This conclusion allows us to view our results in the light of predictions from current theories on sex energy allocation in hermaphrodite and gonochoristic tardigrades. Lastly, we compared the reproductive cycle of these two hermaphroditic species and those of a gonochoristic species belonging to the same genera.

2000 - Tardigrada [Capitolo/Saggio]
Rebecchi, Lorena; A., Guidi; Bertolani, Roberto

In the chapter the various characteristics of tardigrades related to reproduction are reported: anatomy of the male reproductive systems and spermatozoan morphology; maturation pattern and reproductive physiology; fertilization, phylogeny.

1999 - Spermatozoon morphology of three species of Hypsibiidae (Tardigrada, Eutardigrada)and phylogenetic evaluation [Articolo su rivista]
Rebecchi, Lorena; Bertolani, Roberto

The spermatozoan ultrastructure of the gonochoristic eutardigrades Pseudobiotus megalonyx, Ramazzottius tribulosus and Ramazzottius oberhaeuseri (Hypsibiidae, Hypsibiinae) are described and illustrated. The two species of the same genus have very similar spermatozoa, whereas the two genera differ widely in male gamete organization. The testicular spermatozoon of Pseudobiotus has a thread-like configuration and is made up of a small comma-shaped acrosome, a helical nucleus and a tail with terminal tuft; no middle piece or neck and mitochondria were observed. The testicular spermatozoon of Ramazzottius shows an evident rod-shaped acrosome, a cork-screwshaped nucleus and a short tubular middle piece containing a mitochondrial sleeve; moreover, the tail splits terminally into a tuft. Functional considerations and phylogenetic comparisons are made on the basis of sperm morphology.

1999 - Tardigrada [Capitolo/Saggio]
Bertolani, Roberto; Rebecchi, Lorena

In the chapter the various characteristics of tardigrades related to reproduction are reported: modes of reproduction and sex; anatomy of the reproductive systems and germ cell morphology; maturation pattern and reproductive physiology; fertilization; development.

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).

1998 - Evaluation of a secondary sex character in eutardigrades [Articolo su rivista]
Rebecchi, Lorena; D. R., Nelson

Secondary sex characters appear in many heterotardigrades and a limited number of species of eutardigrades. In the eutardigrades Milnesium tardigradum and Pseudobiotus megalonyx, males are considered to always have modified claws on the first pair of legs; the basal branch of both claws in Milnesium and of the inner claw in Pseudobiotus is shaped like a robust hook. We examined one gonochoristic population of both Milnesium tardigradum and of Pseudobiotus megalonyx to evaluate the association of the modified claws with the presence of male germ cells in the testis. Three additional populations of Milnesium tardigradum were examined to provide data on the frequency of the modified claw among all individuals. In both species, molting animals were found with normal claws on the front legs of the old cuticle and modified claws on the front legs of the new cuticle. Examination of the gonad revealed that both species may have males with and without the modified claws. The appearance of the modified claw probably occurs at the last molt, and is used in mating when the male attaches to the female during copulation. Since this secondary sex character is not always present during the life of the tardigrade, but appears after a molt, a sex ratio determined only by the presence of the modified claw is not valid.

1998 - Side effects of the herbicide triasulfuron on collembola under laboratory conditions [Articolo su rivista]
Sabatini, Maria Agnese; Rebecchi, Lorena; C., Cappi; A., Guidi; G., Dinelli; A., Vicari; Bertolani, Roberto

Triasulfuron, a member of the sulfonylureas class of herbicides, was tested under laboratory conditions on the collembolan Onychiurus pseudogranulosus. Pure triasulfuron and commercial formulations at different rates (starting from a dose about 5 times the recommended agricultural rate) were tested separately on one-week old juveniles and adults reared in the laboratory. The persistence of the herbicide at the end of the trials lasting 30 and 60 days was verified by HPLC analysis. Laboratory tests indicated that only the rates exceeding 500 times the soil concentration expected soon after field application of the herbicide directly affected the tested species. (C) 1998 Elsevier Science Ltd. All rights reserved.

1997 - Laboratory and field approaches to evaluate the effects of the herbicide triasulfuron on Collembola. [Abstract in Rivista]
Rebecchi, Lorena; C., Cappi; Sabatini, Maria Agnese; Grazioso, Pasqualina; A., Guidi; A., Vicari; G., Dinelli; Bertolani, Roberto

The effects of the herbicide triasulfuron on Collembola are evaluated in laboratory and field studies.

1997 - Long-term effects of three different continuous tillage practices on Collembola populations [Articolo su rivista]
Sabatini, Maria Agnese; Rebecchi, Lorena; C., Cappi; Bertolani, Roberto; B., Fratello

The long-term effects on Collembola populations of three types of soil tillage (minimum tillage, conventional ploughing to a depth of 25 cm and conventional ploughing to a depth of 50 cm), with four-year rotation (sugar beet, winter wheat, maize, winter wheat), were studied. The responses to rate of N fertilization were also tested. The analysis, which started after 15 years of continuous treatment and extended over a four-year period, revealed very large variations in Collembola abundance in the various years, but found very few significant treatment effects on overall abundances of Collembola. However, analysis carried out at the species level did reveal differences related to tillage; some species were significantly more abundant in plots with minimum tillage and others in plots with conventional ploughing. Moreover, N fertilization affected some species.

1997 - Ultrastructural study of spermiogenesis and the testicular and spermathecal spermatozoon of the gonochoristic tardigrade Xerobiotus pseudohufelandi (Eutardigrada, Macrobiotidae) [Articolo su rivista]
Rebecchi, Lorena

This paper investigates by scanning and transmission electron microscopy spermiogenesis and spermatozoon morphology of the gonochoristic eutardigrade Xerobiotus pseudohufelandi (Macrobiotidae). During spermiogenesis clusters of spermatids are connected by cytoplasmic bridges that persist up to an advanced stage of maturation. Spermiogenesis is characterized by distinctive modifications of the nucleus and by the differentiation of an acrosome, tail and substantial midpiece. Testicular spermatozoa are folded with the hinge located between the head and midpiece, thus resembling a nut-cracker. The head includes a rod-shaped, bilayered acrosome and an elongated, helicoidal nucleus with condensed chromatin. The large kidney-shaped midpiece has hemispherical swellings/ovoid elements surrounding the centriole and an incomplete mitochondrial sleeve. The flagellum contains a ´9 + 2´ axoneme and terminates in a tuft of microtubules. Spermathecal spermatozoa always have linear profiles. The acrosome and nucleus have the same morphological pattern as in testicular spermatozoa, whereas the midpiece is thin and lacks the hemispherical swellings, and the tail is reduced to a short stub. Functional considerations are presented, based upon this different morphology. Moreover, phyletic comparisons are made on the basis of sperm morphology, both for the family Macrobiotidae and the class Eutardigrada.

1996 - Genome size variation in tardigrada [Articolo su rivista]
S., Garagna; Rebecchi, Lorena; A., Guidi

In this paper the genome size evaluation is extended to 8 species of eutardigrades and one species of eterotardigardes in order to obtain a more comphrensive picture of minimun DNA content evolution in the phylum Tardigrada.Genome size and sperm cell morphology are correlated. In the phylum low genome size could be correlated to the high specialization of the phylum.

1996 - Phylogenetic significance of egg shell variation in tardigrades [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena; S. K., Claxton

Freshwater and semiterrestrial tardigrades produce either smooth shelled eggs laid in the exuvium or ornamented eggs laid freely, in contrast to the more primitive marine heterotardigrades which have smooth shelled free-laid eggs. Data are presented on the observation of free-laid ornamented eggs in the primitive non marine heterotardigrade genus Oreella and in a number of eutardigrade genera including Eohypsibius, Amphibolus, Minibiotus and Macrobiotus. These findings have provided a sound basis for the formulation of a hypothesis in which the evolution of the tardigrade egg can be explained by two separate events. The first is the acquisition of ornamentation and the second is the utilization of the exuvium for oviposition with subsequent loss of egg ornamentation. This hypothesis is consistent with new taxonomic arrangements within eutardigrade families and genera (based on character) of the animals) which have led to a more uniform assortment in terms of egg types. This means that egg type (smooth or ornamented) becomes a valid taxonomic character at the generic level.

1996 - Spermatozoan morphology as a character for tardigrade systematics: Comparison with sclerified parts of animals and eggs in eutardigrades [Articolo su rivista]
A., Guidi; Rebecchi, Lorena

The male gamete, a cell widely used for evaluating phylogeny in different animal groups, remains relatively unknown in tardigrades. In this paper the spermatozoa of thirteen species of eutardigrades among four genera and three families is evaluated in order to determine whether sperm morphology can be used as a taxonomic character. Spermatozoa of Amphibolus volubilis and A. weglarskae are very similar and this resemblance is congruent with the remarkable similarity of sclerified parts of the species. In addition, the spermatozoa of eight species of the genus Macrobiotus were examined yielding two groups showing strong intragroup similarities. The first group includes M. pseudohufelandi, M. sandrae, M. macrocalix, M. terminalis and M. joannae, and the second M. richtersi, M. areolatus and M. harmsworthi. Again, these groupings were congruent with those determined by analysis of sclerified structures. In contrast, a marked similarity was found between the spermatozoa of Diphascon (Adropion) scoticum and Platicrista angustata, whereas Diphascon (Diphascon) humicus was appreciably different from both species. Resemblances found in this study between spermatozoa and sclerified body parts suggests that the spermatozoa are suitable characters for use in systematic studies.

1996 - The tardigrades of Emilia (Italy). II. Monte Rondinaio. A multihabitat study on a high altitude valley of the Northern Apennines. [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena

The tardigarde fauna of a small galcila valley on Northern Apennines were investigted. Tardigrades from mosses, lichens, leaf-litter, tuft and water were considered. New tardigrade species were described.

1995 - La lavorazione del terreno e i suoi effetti sulle comunità di microartropodi del suolo [Relazione in Atti di Convegno]
Bertolani, Roberto; Sabatini, Maria Agnese; Rebecchi, Lorena; Grazioso, Pasqualina; B., Fratello

We performed a study on the long-term effects of three types of soil tillage practices and enrichment with two doses of nitrogen fertilizers on soil microartropods. The study carried out in two succesive yeras found no significance differences in the overall population densities of Acarina or Collembola to the species level revealed differences to tillage. it is important that in studeis on agroecosytems at least one of the principal soil micorarthropod taxa be analysed at level species.

1995 - Spermatozoon ultrastructure in two species of Amphibolus (Eutardigrada, Eohypsibiidae) [Articolo su rivista]
Rebecchi, Lorena; A., Guidi

We examined the ultrastructure of the spermatozoa from two species of eutardigrades, gonochoristic Amphibolus volubilis and hermaphoroditic A. weglarkae, by scanning and transmission electron microscopy. The gametes from the two species were morphologically quite similar, each consisting of a short head, neck and tail. The head included a conic, corkscrew-shaped, bilayered acrosome and a cylindrical nucleus with condensed chromatin. The nucleus is surrounded by cytoplasm organized in ovoid elements with an electron-dense core. The neck is very simple, containing a centriole and unmodified mitochondria. The flagellum contains a 9+2 axoneme and terminates in a tuft of between eight and 10 microtubules. The spermatozoa of Amphibolus, like those of the other eutardigrades, are of the modified type, but nonetheless maintain some primitive aspects of the gametes from heterotardigrades.

1995 - Tardigradi della Sardegna e di alcune piccole isole circum-sarde. [Articolo su rivista]
Bertolani, Roberto; A., Guidi; Rebecchi, Lorena

We carried out a faunsitic study on limnic and terrestrial tardigardes from Sardinia and its surroundings samm islands. The study led to the identification of 34 species, including Bryodelphax iohannis, Macrobiosyu biserovi and Macrobiotus serratus, species new for science. Attention is focused on large number of ubiquitous or widespread species.

1994 - Faunistic study in the karst complex of Frasassi (Genga, Italy) [Articolo su rivista]
Bertolani, Roberto; Manicardi, Gian Carlo; Rebecchi, Lorena

La fauna cavernicola del complesso carsico di Frasassi è stata studiata per avere infromazioni dettagliate sulla popolazione aniamle ipogea.

Rebecchi, Lorena; Bertolani, Roberto

We studied the life history of tardigrades with a particular focus on the maturative patterns of the ovary and testis. Specimens collected in nature belonging to four species of one freshwater and two semiterrestrial genera of eutardigrades were examined. The females of all examined species are always iteroparous; they have several maturative cycles with synchronously developing oocytes. Four maturative stages can be distinguished in each ovarian cycle, which is clearly correlated with moulting. In contrast, gametocyte maturation does not seem to be correlated with moulting in males and varies markedly in the considered genera. The testes of adult specimens of Macrobiotus always contain both mature spermatozoa and cells at earlier stages in spermatogenesis (continuous maturation). The testis of Pseudobiotus megalonyx, the only freshwater species examined, shows a gradual increase in spermatozoa that in the end completely fill the gonad (progressive maturation, tied to a semelparous life cycle). The testis in adult males of Amphibolus is either tilled exclusively with spermatozoa or alternatively with immature germinal cells, revealing an iteroparous condition similar to that of the females (cyclical maturation), The different types of gonad maturation in males seem to be correlated with habitat and the capacity to enter cryptobiosis.

1994 - New data on the nuclear DNA content in some species of tardigrades. [Capitolo/Saggio]
Bertolani, Roberto; S., Garagna; Manicardi, Gian Carlo; Rebecchi, Lorena; C. A., Redi

In the chapter the Feulgen DNA content was evlauted in several species of tardigrades. A small genome size and poliploidy was confirmed.

1994 - Ulteriore contributo alla conoscenza dei Tardigradi di Marche e Umbria [Articolo su rivista]
Bertolani, Roberto; Guidetti, Roberto; Rebecchi, Lorena

Studio sulla fauna tardigradologica di Marche ed Umbria. Identificazione di 26 specie una delle quali nuova per la scienza.

1993 - A revision of the Macrobiotus hufelandi group (Tardigrada, Macrobiotidae), with some observations on the taxonomic characters of eutardigrades. [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena

This study considers specimens of Macrobiotus hufelandi C. A. S. Schultze, 1834 collected from Italy and Germany, including the type locality. In addition to a qualitative analysis of the animals and eggs, statistics were performed on the measurements of the sclerified parts of the animals. The data illustrate low intra-species variability in the morphology of both the animals and the eggs. Differences in egg shell morphology, previously attributed to the variability of M. hufelandi, were found to fall into distinct types, related to different animal morphotypes. The data also indicate that M. hufelandi contains several new species: M. macrocalix sp.n., M. sandrae sp.n. and M. terminalis sp.n.; M. hufelandi is redescribed, and a neotype is assigned.

1993 - Tardigradi dell’Appennino umbro-marchigiano [Articolo su rivista]
Bertolani, Roberto; Guidetti, Roberto; Rebecchi, Lorena

Studio faunistico dei tardigradi di Marche ed Umbria. Identificazione di 32 specie di cui una nuova per la scienza.

1992 - Precisazioni su Macrobiotus furciger Murray, 1907, e descrizione di Macrobiotus pilatoi n. sp. (Eutardigrada, Macrobiotidae). [Articolo su rivista]
M. G., Binda; Rebecchi, Lorena

Gli autori forniscono alcuen precisazioni su Macrobiotus furciger Murray 1907 e descrivono la nuova specie Macrobiotus pilatoi che risulta molro simile alla prima tanto da essere confusa con quella. M. furgicer è comune nell'emisfero meridioanle e probabilmente le segnalazioni di quelle specie per le località dell'emisfero settentrionale sono da riferirsi a M. pilatoi.

1992 - Ramazzottius semisculptus, nuova specie di Hypsibiidae (Eutardigrada). [Articolo su rivista]
G., Pilato; Rebecchi, Lorena

Gli autori descrivono una nuova specie di Eutardigrada, Ramazzottius semisculptus, la cui cuticola presenta una scultura (costituita da tubercoli poligonali) appena visibile e limitata alla estremità caudale del corpo; talvolta essa è completamente liscia. Le uova sono deposte libere e sono provviste di piccole sporgenze coniche, lisce, con la base frastagliata.

1991 - A centromeric satellite DNA in the European plethodontid salamanders (Amphibia, Urodela) [Articolo su rivista]
R., Batistoni; I., Nardi; Rebecchi, Lorena; M., Nardone; A., Demartis

A highly repeated satellite DNA (Hy500) located in the centromeric heterochromatin of the European plethodontid salamander Speleomantes (formerly Hydromantes) was studied. The Hy500 family represents about 1% of the Speleomantes supramontis genome and has a major repeating unit of about 500 base pairs, which may have evolved from the progressive amplification of shorter sequences. This centromeric satellite is conserved in all the Speleomantes species, which nevertheless show distinct patterns of chromosomal distribution, which are of relevance as to their phylogenetic relationships.

1991 - First SEM studies on tardigrade spermatozoa. Invertebr. [Articolo su rivista]
Rebecchi, Lorena; A., Guidi

SEM was used to study the external morphology of spermatozoa from two species of Macrobiotus (Eutardigrada). In both species, the spermatozoa consists of 4 parts: (1) a red-shaped acrosome; (2) a helical nucleus; (3) an elongated, swollen middle piece, with lateral vesicles; (4) a tail ending in a tuft of thin and long filaments (probably microtubules). Observations made in vivo and on fresh preparations of sperm cells in toto confirm the images. Moreover, light microscopy findings also showed tahta spermatozoa in the testes and gonoducts have a folded-back acrosomal region, joined to the middle piece. Artificially extracted appeared in some cases straight-headed.

1991 - Karyological analysis on Macrobiotus pseudohufelandi (Tardigrada, Macrobiotidae) and a new finding of a tetraploid population [Articolo su rivista]
Rebecchi, Lorena

Three populations of Macrobiotus pseudohufelandi (Eutardigrada, Macrobiotidae) were studied with karyological techniques not previously utilized for tardigrades. These observations provided detailed information on the karyology of these animals, particularly single chromosome morphology. A diploid amphimictic cytotype and a triploid thelytokous parthenogenetic cytotype were confirmed. In addition, a new tetraploid thelytokous parthenogenetic cytotype was found. Tetraploidy, as defined by chromosome number, was confirmed by DNA-Feulgen content evaluations. Indeed, the minimum DNA content in the tetraploid population is about four fold that in sperm from the diploid population.

Bertolani, Roberto; Rebecchi, Lorena; Beccaccioli, G.

The relationships between the type of reproduction and sexuality and the types of substrates inhabited by semiterrestrial tardigrade populations are reported. Specimens of the genus Ramazzottius (Eutardigrada, Hypsibiidae) were collected from a limited area of hilly to mountainous zones near Modena and Reggio Emilia (Italy). The lichen substrate Xanthoria parietina, typically colonized by R. oberhaeuseri, was the main subject of study, though animals were also extracted from moss cushions. The sex ratio, correlated with the type of reproduction, clearly differed when the lichen or moss samples were collected from tree trunks or limited rocky areas, or came from extensive rocky outcrops. In the former case, the cytological characteristics of oocytes and the absence of males attest to thelytokous parthenogenesis. In the latter, some male specimens were always found, though several samples showed cytological evidence of parthenogenetic and amphimictic females. Differences in the distribution and frequency of the studied tardigrade strains which all utilize passive dispersal mechanisms, notwithstanding substrate and terrain homogeneity, are chiefly due to different modes of reproduction.

1990 - Presenza di Chirocephalus ruffoi Cottarelli e Mura, 1984 (Crustacea, Anostraca) nell'Appennino Tosco-emiliano e descrizione del cariotipo. [Articolo su rivista]
Rebecchi, Lorena; S., Leonardi; Sabatini, Maria Agnese

Viene segnalata la presenza di Chirocephalus ruffoi Cottarelli e Mura, 1984 per l’Appennino tosco-emiliano in due pozze temporanee di alta quota situate sotto il crinale del monte Rondinaio (Modena, Italy). Tale segnalazione rappresenta la seconda in assoluto dopo il locus typicus nell’Appennino calabro-lucano. Gli individui adulti erano presenti da giugno a novembre. Vengono segnalate alcune differenze con il materiale tipico: nella disposizione dei dentelli dell’emipene e nell’aspetto dei processi del corion delle uova. L’analisi cariologica ha messo in evidenza un numero cromosomico aploide di n=12, non diverso da quello delle altre specie congeneri finora studiate. Tuttavia i cromosomi sono risultati tutti metacentrici, a differenza di quelli di C. diaphanus, unica specie per la quale si conosce non solo il numero cromosomico, ma anche il cariotipo.

1988 - New cases of parthenogenesis and polyploidy in the genus Ramazzottius (Tardigrada, Hypsibiidae) and a hypothesis concerning their origin. [Articolo su rivista]
Rebecchi, Lorena; Bertolani, Roberto

Specimens of the genus Ramazzottius Binda and Pilato 1986; were obtained from 2 moss and one lichen sample(s) collected in the Emilian Apennine Mountians. R. tribulosus was only found in one sample, whereas R. oberhaeuseri wa sfound in all three. The first species had only diploid specimens, with 6 bivalents during the first meiotic division; the second had only females showing various polyploid cytotypes in addition to the diploid bisexual cytotype cited for this area. One of the triploid and the tetraploid cytotyoes were charactetized by the presence of univalents at oocyte metaphase, Though the large number of cytotypes found in a single sample may be attributed to chance, it is better explained by an in loco origin, at least in soem cases.

1988 - The tardigrades of Emilia (Italy). I. Rossena. [Articolo su rivista]
Bertolani, Roberto; Rebecchi, Lorena

The following species are found in a moss collected at Rossena (Reggio Emilia, Italy): Cornechinioscus lobatus, Echinicus trisetosus, Macrobiotus csotiensis, M. areolatus, M. cf. hufelandi, Doryphoribius flavus, Ramazzottius oberhaeuseri and a new species, Ramazzottius tribulosus, very similar to R. oberhaeuseri, but clearly dostinguishable by its cone -shaped egg processes.