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Roberto GUIDETTI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Biologia

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Pubblicazioni

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
abstract

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 - A new, simplified, drying protocol to prepare tardigrades for scanning electron microscopy [Articolo su rivista]
Camarda, Daniele; Massa, Edoardo; Guidetti, Roberto; Lisi, Oscar
abstract

A new protocol for preparation of tardigrades for scanning electron microscope (SEM) analysis is proposed. The more conventional protocols require various steps and a long time to obtain good drying of water bears, together with specific and uncommon instruments (i.e., critical point dryer) or highly volatile toxic compounds (i.e., hexametildisilazane). The new protocol can be performed using few and simple instruments and materials, all easily accessible, and produces a high yield in terms of dried animals in excellent condition for the observation of external morphological structures with SEM. The acquired data exhibit considerable promise, and the proposed methodology shows potential for application to other meiofaunal groups, including small arthropods, nematodes, and rotifers. RESEARCH HIGHLIGHTS: Cheap, safe, and fast new method for Tardigrada preparation for SEM. With the new protocol, the number of animals required for SEM studies is minimized. New protocol is potentially applicable to the study of other meiofaunal soft-bodied taxa.

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
abstract

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.
abstract

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 - Tardigrade diversity and community composition across Norwegian boreal forests [Articolo su rivista]
Guidetti, R; Jönsson, Ki; Kaczmarek, L; Meier, T; Speed, Jdm; Presto, T; Stur, E; Topstad, L; Cesari, M; Roszkowska, M; Zawierucha, K; Hassel, K; Ekrem, T
abstract

Tardigrades are common in many terrestrial environments and habitats. Although little is known on their ecological preferences, previous studies found diversity and community composition significantly affected by various variables. This study associated tardigrade species' records with climatic variables, forest type, and substrate categories exploring tardigrade diversity and species communities to find associations with ecological characteristics of Norwegian forests.A total of 17 473 specimens were identified, encompassing 131 species (including putatively new species) from 305 samples of different substrates (leaf litter, bryophytes, and lichens). Bryophytes and lichens of samples were classified according to the main species, and growth form, and associated with tardigrade species and sample metadata. Tardigrade species' richness was related to climatic variables and forest type, increased with precipitation, decreased with summer temperature, and not varied with precipitation seasonality. Although there was an unbalanced representation of substrate categories in the different forest types, some tendencies were detectable. Mixed oak and birch forests reached the highest species' richness. Tardigrade community composition varied between substrate categories and, to a lesser degree, between forest types, but not with climatic variables. Our study highlights the importance of large-scale variables on tardigrade diversity, and substrate categories for tardigrade community composition.

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
abstract

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
abstract

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 - Identification of predatory arthropods of the invasive Halyomorpha halys through molecular gut content analysis [Articolo su rivista]
Bulgarini, G.; Piemontese, L.; Guidetti, R.; Cesari, M.; di Bella, E.; Maistrello, L.
abstract

Halyomorpha halys (Stål, 1855) is an invasive agricultural pest in North America and Europe. Most of the information on H. halys predators in invaded areas comes from North America. This work focused on the molecular identification of arthropod predator species capable of feeding on H. halys in northern Italy. Predatory arthropods were collected in the field in four urban parks using the tree-beating technique. A real-time PCR workflow was applied to detect H. halys DNA from the gut content of predators. Of the 190 predator individuals analysed, 46 were positive for H. halys DNA and belonged to 10 insect taxa (1 Dermaptera, 3 Coleoptera, 2 Hemiptera and 4 Orthoptera) and six arachnid taxa (2 Opiliones and 6 Araneae). The integration of gut content analysis with laboratory bioassays and field observations allows the identification of a greater number of predators and therefore a better understanding of how the invaded ecosystem is responding to the introduction of a new species, given that samples are taken from the invaded environment itself. Therefore, the gut content analysis provides essential elements for conservation biocontrol in integrated pest management programmes.

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
abstract

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
abstract

: 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.
abstract

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
abstract

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
abstract

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
abstract

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.

2022 - The morphological diversity within a species can obscure the correct identification [Articolo su rivista]
Lo Brutto, Sabrina; Schimmenti, Eugenia; Iaciofano, Davide; Lubinevsky, Hadas; Cesari, Michele; Guidetti, Roberto
abstract

Critical points of various diagnostic characters and a paucity of information relating to the geographical distribution of several marine species can hinder real species delimitation, particularly if they are supposed to be cosmopolitan. Such constraints characterize many amphipod species and are mainly due to the variation in morphological characters during growth. Specifically, the benthic filter-feeding corophiid Cheiriphotis mediterranea Myers, 1983 displays different shapes for the male gnathopod 2 as it grows. This variation has hitherto never been described but an extensive sampling has provided us with the opportunity of studying it in detail. More than six thousand individuals, belonging to C. mediterranea, were collected along the Israeli coast during a five-year soft-bottom monitoring survey, comprising 282 sites at a depth between 4 and 37 m. One of the objectives of this study was to integrate the original description of C. mediterranea with the identification of definite six morphotypes, which alternate during growth. A mtDNA Cytochrome c oxidase subunit I sequence was obtained, representing the first molecular datum for the genus Cheiriphotis. The results offer the possibility of including variable morphological characters and molecular sequences in the identification process. Throughout the survey, C. mediterranea was confirmed to be one of the most abundant five species in the study area, in particular in Haifa Bay, where the environmental conditions are stressful. Barely-known abundance and distribution notes relating to this species are reported in this paper; a putative role of bioindicator is also suggested.

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.
abstract

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 - Multi-marker dna metabarcoding reflects tardigrade diversity in different habitats [Articolo su rivista]
Topstad, L.; Guidetti, R.; Majaneva, M.; Ekrem, T.
abstract

Like meiofauna in general, tardigrades are often neglected in ecological and environmental surveys. Tardigrades occur in all parts of the world, from deep marine sediments to alpine environments, and are present in most ecosystems. They are therefore potentially good candidates for biomonitoring programs. However, sampling of these minute animals is both tedious and time-consuming, impeding their inclusion in large-scale ecological surveys. In this study we argue that using a multi-marker metabarcoding approach on environmental DNA (eDNA) partly can overcome this barrier. Samples of moss, lichens, and leaf litter were investigated both by morphologybased methods and DNA metabarcoding, and the results were compared in terms of tardigrade diversity and community composition of the sampled microhabitats. DNA metabarcoding using three markers detected more species of tardigrades than identification by morphology in most samples. Also, metabarcoding detected the same community differences and microhabitat distribution patterns as morphology-based methods. In general, metabarcoding of litter samples was unreliable, with only one out of three markers consistently amplifying and detecting tardigrades. The low availability of tardigrade reference sequences in public databases restricts the taxonomic resolution in eDNA surveys, but this impediment is partly circumvented by utilizing multiple markers.

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
abstract

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.
abstract

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.
abstract

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
abstract

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
abstract

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 - 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
abstract

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

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

2020 - OSITOS DE AGUA: generalidades, origen, evolución y su estado de conocimiento en México [Articolo su rivista]
ANDRÉS PÉREZ PECH, Wilbert; Guidetti, Roberto; ANGUAS ESCALANTE, Abril; QUERUBIN CUTZ POOL, Leopoldo
abstract

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

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.
abstract

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.

2020 - Two new species of Tardigrada from moss cushions (Grimmia sp.) in a xerothermic habitat in northeast Tennessee (USA, North America), with the first identification of males in the genus Viridiscus [Articolo su rivista]
Nelson, Diane R.; Adkins Fletcher, Rebecca; Guidetti, Roberto; Roszkowska, Milena; Grobys, Daria; Kaczmarek, Łukasz
abstract

Background. The phylum Tardigrada consists of over 1,300 species that inhabit terrestrial, freshwater and marine environments throughout the world. In terrestrial habitats they live primarily in mosses, lichens, leaf litter and soil, whereas tardigrades in freshwater and marine environments are mainly found in sediments and on aquatic plants. More than 65 species have been previously reported in the state of Tennessee, USA. Methods. Tardigrades present in moss cushions (Grimmia sp.) collected from a xerothermic habitat on the East Tennessee State University campus, Johnson City, TN, USA, were extracted, mounted on slides, identified, and counted. Additional samples of fresh dried moss were used for integrative analyses, including morphological analysis with phase contrast (PCM) and scanning electron microscopy (SEM), as well as molecular analyses of COI, 18S rRNA, 28S rRNA, and ITS-2 of the Macrobiotus and Milnesium species. Results. Five species were found, including two species new to science: Viridiscus miraviridis sp. nov. and Macrobiotus basiatus sp. nov. Viridiscus miraviridis sp. nov. differs from other members of the genus mainly by having a different type of dorsal cuticle and some other, more subtle, morphometric characters. In addition to the two new species, Viridiscus perviridis and Viridiscus viridissimus were present, and males of Vir. viridissimus were found for the first time, the first record of males in the genus Viridiscus. Macrobiotus basiatus sp. nov. is most similar to Macrobiotus nelsonae, but it differs from Mac. nelsonae mainly by the stylet supports being situated in a more anterior position, shorter and narrower egg processes, and a smaller number of areoles around the egg processes. Moreover, the identification of Milnesium inceptum was confirmed as the first record for the USA by analysis of COI.

2019 - A closer look at the antennae of the invasive Halyomorpha halys: fine structure of the sensilla [Articolo su rivista]
Ibrahim, Aya; Giovannini, Ilaria; Anfora, Gianfranco; Valerio ROSSI STACCONI, Marco; Malek, Robert; Maistrello, Lara; Guidetti, Roberto; Romani, Roberto
abstract

The brown marmorated stink bug, Halyomorpha halys, is an invasive agricultural and urban pest capable of feeding on over 100 species of host plants. The antennae of this bug play an important role not only in detecting food and mates but also in short-range location of conspecifics when aggregating for diapause. The morphology and distribution of antennal sensilla of H. halys were investigated at an ultrastructural level using scanning and transmission electron microscopy approaches. Adults have 5-segmented antennae, made up of a scape, a 2-segmented pedicel and two flagellomeres, while 5th instar nymphs have shorter, 4-segmented antennae, with only one pedicel segment. Five types of sensilla are distinguished, based on their shape, length and basal width and the presence of basal socket and pores: sensilla basiconica (types A, B, C, D and E), sensilla coeloconica, sensilla trichoidea and sensilla chaetica (types A and B). Sexual dimorphism was not observed in this species, with respect to the morphological features and abundance of sensilla. The most abundant sensory structures are sensilla trichoidea showing characteristics typical of olfactory sensilla, whereas the least abundant are sensilla coeloconica which may be involved in thermo- and hygro-reception. Basiconic sensilla type A are solely identified on the antennae of 5th instar nymphs, where they presumably have a dual mechanosensorygustatory role. The putative function of the remaining sensilla are herein discussed.

2019 - Actual checklist of Tardigrada species [Banca dati]
Degma, Peter; Bertolani, Roberto; Guidetti, Roberto
abstract

More than one thousand Tardigrada species were included in the published checklist (Guidetti, R. & Bertolani, R. 2005. Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification. Zootaxa, 845, 1–46.) plus the additions and corrections to this checklist (Degma, P. & Guidetti, R. 2007. Notes to the current checklist of Tardigrada. Zootaxa, 1579, 41–53.). For practical reasons, we have joined these two papers (without comments added to particular taxa as well as without references published in these papers) into an accurate combined version of the checklists. We incorporated all taxonomical novelties in the current edition of the Checklist even if they were published just online. Then, following the International Code of Zoological Nomenclature, we corrected the year of a taxon description according to its print edition. This checklist is free for all users, but utilization of it requires the citation of the two original papers. This checklist is the platform for occasional upgrades (date of latest upgrade is in the title). The changings respect to the previous version of the checklist have yellow background. If you also use these changes, please also cite this checklist (please also cite this checklist (Degma, P. & Guidetti, R. Actual checklist of Tardigrada species. DOI: 10.25431/11380_1178608. Accessed date). Please write us if you find any mistake or missing data in this checklist. Your help in its improvement will be acknowledged.

2019 - CMOS and 3D Printing for NMR Spectroscopy at the Single Embryo Scale [Articolo su rivista]
Grisi, Marco; Montinaro, Enrica; Vincent, Franck; Pethö, Laszlo; Letizia, Maria Cristina; Volpe, Beatrice; Harris, Nicola; Beck, Armin; Guidetti, Roberto; Gijs, Martin; Michler, Johann; Brugger, Jürgen; Boero, Giovanni
abstract

New combination of CMOS and 3D printing technologies achieves state-of-the-art sensitivity for the NMR studies of nanoliter and subnanoliter living biological entities.

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

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
abstract

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.
abstract

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
abstract

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

2018 - 3D printed microchannels for sub-nL NMR spectroscopy [Articolo su rivista]
Montinaro, E.; Grisi, M.; Letizia, M. C.; Petho, L.; Gijs, M. A. M.; Guidetti, R.; Michler, J.; Brugger, J.; Boero, G.
abstract

Nuclear magnetic resonance (NMR) experiments on subnanoliter (sub-nL) volumes are hindered by the limited sensitivity of the detector and the difficulties in positioning and holding such small samples in proximity of the detector. In this work, we report on NMR experiments on liquid and biological entities immersed in liquids having volumes down to 100 pL. These measurements are enabled by the fabrication of high spatial resolution 3D printed microfluidic structures, specifically conceived to guide and confine sub-nL samples in the sub-nL most sensitive volume of a single-chip integrated NMR probe. The microfluidic structures are fabricated using a two-photon polymerization 3D printing technique having a resolution better than 1 μm3. The high spatial resolution 3D printing approach adopted here allows to rapidly fabricate complex microfluidic structures tailored to position, hold, and feed biological samples, with a design that maximizes the NMR signals amplitude and minimizes the static magnetic field inhomogeneities. The layer separating the sample from the microcoil, crucial to exploit the volume of maximum sensitivity of the detector, has a thickness of 10 μm. To demonstrate the potential of this approach, we report NMR experiments on sub-nL intact biological entities in liquid media, specifically ova of the tardigrade Richtersius coronifer and sections of Caenorhabditis elegans nematodes. We show a sensitivity of 2.5x1013spins/ Hz1/2on1H nuclei at 7 T, sufficient to detect 6 pmol of1H nuclei of endogenous compounds in active volumes down to 100 pL and in a measurement time of 3 hours. Spectral resolutions of 0.01 ppm in liquid samples and of 0.1 ppm in the investigated biological entities are also demonstrated. The obtained results may indicate a route for NMR studies at the single unit level of important biological entities having sub-nL volumes, such as living microscopic organisms and eggs of several mammalians, humans included.

2018 - Environmental Adaptations: Encystment and Cyclomorphosis [Capitolo/Saggio]
Guidetti, Roberto; Møbjerg, Nadja
abstract

Stressful environmental conditions generally limit animal survival, growth, and reproduction and may induce dormancy in the form of various resting stages. Tardigrades represent one of a few animal phyla in which different forms of dormancy are frequently encountered. One of these forms, cryptobiosis, a quick response to sudden changes in the environment, has gained a great deal of attention, whereas much less is known of the slower emerging form of dormancy, diapause. In this review we present the current knowledge of diapause in tardigrades. Diapause in tardigrades, represented by encystement and cyclomorphosis, is likely controlled by exogenous stimuli, such as temperature and oxygen tension, and perhaps also by endogenous stimuli. These stimuli initiate and direct successive phases of deep morphological transformations within the individual. Encystment is characterized by tardigrades that lie dormant—in diapause—within retained cuticular coats (exuvia). The ability to form cysts is likely widespread but presently only confirmed for a limited number of species. In tardigrades, cyclomorphosis was first reported as a characteristic of the marine eutardigrade genus Halobiotus. This phenomenon is characterized by pronounced seasonal morphological changes and in Halobiotus involves stages with an extra protecting cuticle. Cyst formation in moss-dwelling limnic species may also occur as part of a seasonal cyclic event and can thus be viewed as part of a cyclomorphosis. Therefore, whereas diapause generally seems to be an optional response to environmental changes, it may also be an obligate part of the life cycle. The evolution of encystment and cyclomorphosis finds its starting point in the molting process. Both phenomena represent an adaptation to environmental constraints. Notably, the evolution of diapause is not necessarily an alternative to cryptobiosis, and some tardigrades may enter both forms of dormancy. The simultaneous occurrence of several adaptive strategies within tardigrades has largely increased the resistance of these enigmatic animals toward extreme environmental stress.

2018 - Genetic Diversity of Halyomorpha halys (Hemiptera, Pentatomidae) in Korea and Comparison with COI Sequence Datasets from East Asia, Europe, and North America [Articolo su rivista]
Lee, Wonhoon; Guidetti, Roberto; Cesari, Michele; Gariepy, T. D.; Park, Yong-Lak; Park, Chang-Gyu
abstract

The brown marmorated stinkbug, Halyomorpha halys, is an invasive insect pest in North America and Europe that attacks crop species and causes substantial economic damage. To evaluate the genetic diversities and distributions of different H. halys populations in East Asia, North America, and Europe, COI sequences obtained from 79 new specimens from Korea and 10 from the USA were compared with 725 existing COI sequences. In total, 45 haplotypes were detected in populations from 10 countries. Sixteen haplotypes from Korea (H34–H49) and 2 from the USA (H50 and H51) were novel. Korean populations exhibited the 2nd highest diversity among the 10 countries, with only Greece exhibiting higher diversity. Haplotype H22 was prominent in Korea, H1 was prominent in China, Greece, Hungary, Italy, Canada, and USA, and H3 was prominent in France and Switzerland. Of the 18 haplotypes found in Korea, 1 was shared with China (H2) and 1 with Greece (H22). Haplotype diversity patterns showed that Korean populations were genetically distinct from populations in China, Europe, and North America. This suggested that populations in Europe and North America arose through multiple invasions from China and that (with the exception of Greece), Korean populations did not spread to other countries. This study represents a comprehensive analysis of H. halys populations in Korea and places these populations in a global context that includes other native populations in East Asia and invasive populations in Europe and North America.

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
abstract

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
abstract

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 - Paleontology and Molecular Dating [Capitolo/Saggio]
Guidetti, Roberto; Bertolani, Roberto
abstract

Few fossil tardigrade records are known, probably due to their minute size and absence of consistent hard body parts. A possible but not certain tardigrade ancestor was found as an “Orsten”-type secondarily phosphatized fossil from the Middle Cambrian limestone. It is characterized by only three pairs of legs as a possible consequence of a plesiomorphic anameric ontogeny that led to four leg segments in the extant tardigrades. Its other characters, related to claw and cuticle morphologies, resemble those of heterotardigrades. The first findings of sure tardigrades come from Cretaceous amber, in which the species Beorn leggi and Milnesium swolenskyi were found. These species, and especially the latter, evidence that more than 90 million years ago, “modern” tardigrades already existed, and only few morphological differences, mainly related to morphometric tracts, occur with respect to known modern species. Quaternary subfossils of tardigrades have been found with palynological studies during paleoecology researches. They have been recorded mainly in polar regions and classified within the non-pollen palynomorphs category. Cuticular remains of animals and eggs can persist in the substrates for very long time, so tardigrades can be used as paleoclimatic bioindicators, but despite their potentiality, a few studies have focused to find these organisms in microfossil samples and in sediments for paleoecological studies. For estimating the dates of phylogenetic events related to the origin of tardigrades and/or their evolutionary lineages, molecular clock analyses have been used. Molecular data indicate that the origin of the phylum should be posed during or before the Cambrian period, placing the origin of Tardigrada in marine environment and their terrestrialization not before that of other ecdysozoans.

2018 - Tardigrade Taxa [Capitolo/Saggio]
Degma, Peter; Guidetti, Roberto
abstract

The list of tardigrade taxa up to a sub-generic level, with the type species of each genus and the numbers of lower taxa in each taxon (known in the end of June 2018) is here presented together with the main environments in which taxa were found. Each listed taxon is characterized by characters common to all lower taxa using predominantly most recent taxa definitions. Taxa definitions were eventually adjusted using original descriptions. This review of tardigrade taxa underlines as taxonomy and systematics of the phylum are continuously updated by researchers and new species are constantly discovered. Currently, there are 1265 species (2 of them fossil) of Tardigrada belonging to 129 genera and 28 families including one fossil.

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
abstract

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
abstract

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 - Attraction of Halyomorpha halys (Hemiptera: Pentatomidae) haplotypes in North America and Europe to baited traps [Articolo su rivista]
Morrison, William R.; Milonas, Panos; Kapantaidaki, Despoina Evr; Cesari, Michele; DI BELLA, Emanuele; Guidetti, Roberto; Haye, Tim; Maistrello, Lara; Moraglio, Silvia T.; Piemontese, Lucia; Pozzebon, Alberto; Ruocco, Giulia; Short, Brent D.; Tavella, Luciana; Vétek, Gábor; Leskey, Tracy C.
abstract

Halyomorpha halys is a global invasive species, native to Southeast Asia, that is threatening agriculture in invaded regions. Our objectives were to: 1) establish the attractiveness of semiochemical stimuli paired with feld-deployed traps in Europe (Greece, Hungary, Italy, and Switzerland), compared with Maryland, USA, and 2) identify H. halys haplotypes recovered from traps at each location. We found qualitatively identical patterns of capture between sites located across Europe and in Maryland, USA. In both regions, captures of H. halys adults indicated a synergistic response to traps baited with the two component H. halys aggregation pheromone, and pheromone synergist, methyl (2E, 4E, 6Z)-decatrienoate when compared with either individually. Haplotype diversity in Europe based on trapped specimens was much greater than the USA, with fve new haplotypes described here, probably indicating ongoing invasion and re-introduction of H. halys. By contrast, a single, previously identifed haplotype was trapped in Maryland, USA, representing a single introduction. All H. halys haplotypes responded to each semiochemical in apparent proportion to their frequency in the overall population based on independently derived information from prior work. Taken together, these data suggest that pheromone-based technology will be of global utility for the monitoring of this important invasive species

2017 - Comparative analyses of Bertolanius species (Eohypsibiidae; Eutardigrada) with the description of Bertolaniusbirnae sp. nov. from northern polar regions [Articolo su rivista]
Hansen, Jesper Guldberg; Kristensen, Reinhardt Møbjerg; Bertolani, Roberto; Guidetti, Roberto
abstract

Species of the genus Bertolanius (Eohypsibiidae, Eohypsibioidea) are morphologically very similar and can be distinguished by only minute differences. During faunal surveys in Arctic areas (Disko I., Greenland; Uummannaq, Greenland; Abisko, Sweden), the four species Bertolanius nebulosus, Bertolanius smreczynskii, Bertolanius weglarskae and Bertolanius birnae sp. nov. were found. Bertolanius nebulosus and Bertolaniussmreczynskii have been compared with their paratypes, and type material of other species of Bertolanius has been considered for comparison within the genus. Bertolaniusbirnae sp. nov. possesses a unique combination of characters (i.e., an evident anterior band of teeth, the median ridge of the buccal armature consisting of two large teeth instead of one, absence of eyes, and eggs with conical processes without areolation around their bases) that is not seen in any other species in the genus, though these characters may be present in other combinations within the genus. Our revision and analytical comparison among these Bertolanius species adds new and important information on claw, buccal armature, egg surface morphologies as well as the encystment process of the species. We also provide a taxonomic key to aid the identification of Bertolanius species. The new discoveries of Bertolanius species from northern polar regions further support the hypothesis that the distribution of Eohypsibioidea is restricted to the Holarctic in the Arctic and Periarctic areas, or in alpine areas between 550 and 2400 m a.s.l. of more temperate regions.

2017 - Doryphoribius chetumalensis sp. nov. (Eutardigrada: Isohypsibiidae) a new tardigrade species discovered in an unusual habitat of urban areas of Mexico [Articolo su rivista]
Pérez-pech, Wilbert Andrés; Anguas-escalante, Abril; Cutz-pool, Leopoldo Querubin; Guidetti, Roberto
abstract

A new species, Doryphoribius chetumalensis, is described from specimens collected in the city of Chetumal (Quintana Roo state, Mexico). The species was found in a new and unusual habitat for urban tardigrades, i.e. the soil sediment accumulated on the border of streets. This discovery shows that tardigrades can live in this habitat, demonstrating once again the wide capacity of this taxon to tolerate adverse habitats, and to survive in environments with high anthropogenic impact. Doryphoribius chetumalensis sp. nov. differs from all the other species of the genus in having enlarged and wide bulbous base of the claws. Within Doryphoribius, it belongs to the zappalai group, and differs from the species in this group, not only in the claw shape, but also by the orange body colour, the smooth cuticle, the absence of a tooth in the wall of the buccal ring, and the absence of lunules under the claws. This is the first record of tardigrades, identified to species level, in Quintana Roo state. A taxonomic key of the Doryphoribius genus is also presented.

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
abstract

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 - NMR spectroscopy of single sub-nL ova with inductive ultra-compact single-chip probes [Articolo su rivista]
Grisi, Marco; Vincent, Franck; Volpe, Beatrice; Guidetti, Roberto; Harris, Nicola; Beck, Armin; Boero, Giovanni
abstract

Nuclear magnetic resonance (NMR) spectroscopy enables non-invasive chemical studies of intact living matter. However, the use of NMR at the volume scale typical of microorganisms is hindered by sensitivity limitations, and experiments on single intact organisms have so far been limited to entities having volumes larger than 5 nL. Here we show NMR spectroscopy experiments conducted on single intact ova of 0.1 and 0.5 nL (i.e. 10 to 50 times smaller than previously achieved), thereby reaching the relevant volume scale where life development begins for a broad variety of organisms, humans included. Performing experiments with inductive ultra-compact (1 mm2) single-chip NMR probes, consisting of a low noise transceiver and a multilayer 150 μm planar microcoil, we demonstrate that the achieved limit of detection (about 5 pmol of 1H nuclei) is sufficient to detect endogenous compounds. Our findings suggest that single-chip probes are promising candidates to enable NMR-based study and selection of microscopic entities at biologically relevant volume scales.

2017 - Use of substrate-borne vibrational signals to attract the Brown Marmorated Stink Bug, Halyomorpha halys [Articolo su rivista]
Mazzoni, Valerio; Polajnar, Jernej; Baldini, Marta; Rossi Stacconi, Marco Valerio; Anfora, Gianfranco; Guidetti, Roberto; Maistrello, Lara
abstract

Despite the increasing number of studies on the use of acoustic stimuli to control agricultural pests, this approach is still theoretical. Many insect pests, in particular hemipterans, use vibrational signals for mating communication and therefore the application of a control strategy based on acoustic interference is a promising option. The Brown Marmorated Stink Bug, Halyomorpha halys, is causing severe economic damage on many crops in the USA and Italy. We tested a female vibrational signal, Female Signal 2 (FS2), to attract males in different settings, such as natural substrate, arenas and a cage representing an acoustic trap. We used video tracking analysis and described the vibrational amplitude field around the individuals to study the male behaviour. We found that FS2 can attract more than 50% of males to the source point and has a strong “loitering” effect on searching males that tend to remain in the stimulated area. We concluded that FS2 exhibits good attractiveness to H. halys males and that its potential use as a tool integrated in the currently existing pheromone traps should be tested in the field.

2016 - An overview on the invasive pest Halyomorpha halys in Northern Italy: biology, field monitoring and IPM approaches [Abstract in Atti di Convegno]
Maistrello, Lara; Caruso, S.; Costi, Elena; Vaccari, G.; Bortolini, Sara; Guidetti, Roberto; Bortolotti, P.; Nannini, R.; Casoli, L.; Vergnani, S.; Montermini, A.
abstract

A few years after the first appearance in Italy in 2012, the Asian Brown Marmorated Stink Bug (BMSB) Halyomorpha halys (Heteroptera, Pentatomidae) became a serious pest of fruit crops in Northern regions, causing up to 80% damage in some areas of Emilia Romagna pear orchards in 2015. In this Region the network between the University and the Plant Protection Services of Modena and Reggio Emilia allowed to act sinergically in carrying out a multidisciplinary applied research project on this invasive pest. A citizen science survey allowed to obtain the updated BMSB spread map and the DNA analysis of collected samples showed that Italy has the highest haplotype diversity beyond native Asian population, indicating multiple invasion pathways. A detailed life table study showed that BMSB performs two generations/year with overlapping adult and juvenile stages during summer, and has a remarkable invasive potential due to its high rate of increase and specific behavioural features. A three years monitoring program in orchards and adjacent uncultivated areas assessed various aspects of the insect biology and the applicability of active and passive monitoring techniques to estimate the presence, abundance, and seasonality of BMSB populations in the field. Presently, crop management relies on insecticides, however different IPM approaches are being evaluated, that include a survey on native natural antagonists potential, a behavior-based management of orchards boards and hedges and the use of exclusion netting.

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.
abstract

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
abstract

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 - Halyomorpha halys in Italy: first results of field monitoring in fruit orchards [Articolo su rivista]
Maistrello, Lara; Costi, Elena; Caruso, S.; Vaccari, G.; Bortolotti, P.; Nannini, R.; Casoli, L.; Montermini, A.; Bariselli, M.; Guidetti, Roberto
abstract

The invasive pest Brown Marmorated Stink Bug (BMSB) Halyomorpha halys (Heteroptera, Pentatomidae) was detected for the first time in Italy in September 2012 in Modena province (Northern Italy) during an insect collection for educational purposes. A survey performed in 2013 allowed to detect its presence in Emilia Romagna, Lombardy and Piedmont regions. In 2014, in the provinces of Modena, Reggio Emilia and Bologna a periodical active field monitoring was performed using tree beating, sweep-net and visual observations in selected orchards and vineyards, recording numbers of BMSB adults and nymphs, and of other Heteroptera. Besides, fruit injury and crop loss were recorded at harvest. Partial results from field data obtained between April and July 2014 are presented, indicating that BMSB is already becoming an important pest of fruit orchards and that special attention should be deserved to monitor its spread all over the region and the whole Italian country.

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
abstract

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
abstract

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 - Monitoraggio in campo e danni della cimice aliena Halyomorpha halys in Emilia Romagna: da minaccia a problema concreto. [Relazione in Atti di Convegno]
Maistrello, Lara; Vaccari, Giacomo; Bortolini, Sara; Costi, Elena; Guidetti, Roberto; Bortolotti, Paolo; Caruso, Stefano; Nannini, Roberta; Montermini, Anselmo; Casoli, Luca
abstract

A few years after the first appearance in Italy of the Asian brown marmorated stink bug Halyomorpha halys (Heteroptera, Pentatomidae), established in 2012, a monitoring program was performed in the provinces of Modena and Reggio Emilia in order to assess various aspects of the insect biology and the applicability of active and passive monitoring techniques to estimate the presence, abundance, and seasonality of H. halys populations in the field. In 2015, a study was carried out to compare the different monitoring techniques, which included visual survey, tree beating, sweep-netting, and the use of traps baited with aggregation pheromone. The survey was performed both in orchards and in the adjacent uncultivated areas. Our observations indicate that the pest has two generations with overlapping of different developmental stages between May and September. The preliminary elaborations show that in surveyed areas the populations are progressively increasing and cause significant yield loss, especially in the border rows of the fields. These results are useful in the view to organize more targeted monitoring activities and provide constructive suggestions for management strategies.

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
abstract

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
abstract

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
abstract

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

2016 - Potential use of vibrational signals to manage the Brown Marmorated Stink Bug, Halyomorpha halys, a new invasive agricultural pest [Abstract in Atti di Convegno]
Mazzoni, Valerio; Anfora, Gianfranco; Rossi Stacconi, Marco Valerio; Polajnar, Jernej; Baldini, Marta; Guidetti, Roberto; Maistrello, Lara
abstract

The Brown Marmorated Stink Bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) is a polyphagous insect that can cause severe economic damage on different crops. In Italy this species was first recorded in the region Emilia Romagna in 2012 and in few years it spread in most of the Northern and part of the Central Italian regions. As well as in other stink bugs the mid-short range mating communication is based on the exchange of vibrational signals (duet) between sexes that is the key of pair formation, courtship and copula. During the duet females are stationary and males actively search on the plant. In other pentatomids the directionality of the searching in males, given by female attractive signals, has been demonstrated. We hypothesize that the same principle works also in BMSB and thus it would be possible to use the female calling signal to attract males towards precise source sites (i.e. artificial shakers). If confirmed, this would represent a potential implementation to the traps commonly used for BMSB monitoring, by providing an additional stimulus to enlarge the amount of captured bugs. To assess whether the female calling signal could be employed to capture males we performed different bioassays with males stimulated with either natural and synthetic vibrational signals, in different scenarios (i.e., net cages, round arenas, potted plants). Vibrational signals were recorded with laser vibrometer and a video analysis software was used to study the behavioural responses. Our experiments demonstrated the attractive effect of the female signal in all performed tests. On average, more than 50% of the tested males were attracted to vibrating sources. In addition, we also found a significant increase of walking speed, walked distance and time spent in stimulated areas. These results show promise for developing more efficient trapping techniques against H. halys in both agricultural and urban environments.

2016 - Primer Registro Genérico de tardígrades, habitantes del área urbana de Chetumal Quintana Roo, México [Articolo su rivista]
Pérez Pech, A. Wilbert; Cutz Pool, Q. Leopoldo; Guidetti, Roberto; Blanco Piñón, Alberto
abstract

This study provides the first records of tardígrades for the state of Quintana Roo, Mexico specifically from the urban area of Chetumal. Are given descriptions of the genera Doryporibius, Macrobiotus and Echiniscus. Are documented the biotopes in what were tardigrade, this study is a pioneer describe tardigrades collected in the ground above asphalt road in an urban area. It discusses its richness and geographical distribution in the country.

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
abstract

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 - 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
abstract

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
abstract

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
abstract

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 - A task force to develop alternative strategies against emerging alien pests: Drosophila suzukii and Halyomorpha halys in Northern Italy. [Poster]
Caruso, Stefano; Vaccari, Giacomo; Guidetti, Roberto; Costi, Elena; Maistrello, Lara
abstract

The Spotted Wing Drosophila (SWD), Drosophila suzukii, and the Brown Marmorated Stink Bug (BMSB), Halyomorpha halys, are two polyphagous invasive pests native to east Asia, recently introduced in Northern Italy. SWD is a pest of soft-skinned fruits such as cherries and berries, whereas BMSB can attack many fruits, and horticultural and ornamentals crops. After their introduction, specific field monitoring campaigns were conducted and allowed to recognize that both pests have increasingly expanding distribution ranges and are seriously threatening cherry and other fruit orchards. Studies are being performed on the biology and potential for biological control by autochtonous antagonists in the newly invaded areas. For both targets, applied research projects are being performed, aimed at: a) the implementation of monitoring methods to improve capture efficiency and selectivity, together with damage identification and evaluation, and b) the development of sustainable management strategies based on behaviour manipulation, such as mass trapping, attract-and-kill, push-pull, pest exclusion (anti-insect nets) and verify the potential of autochtonous antagonists.

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
abstract

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
abstract

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 - Halyomorpha halys in Emilia, prime risposte dal monitoraggio [Articolo su rivista]
Bortolotti, Paolo; Caruso, Stefano; Nannini, Roberta; Vaccari, Giacomo; Casoli, Luca; Bariselli, Massimo; Costi, Elena; Guidetti, Roberto; Maistrello, Lara
abstract

OSSERVAZIONI IN CAMPO CONDOTTE NEL 2013-2014. Dopo la sua comparsa in Italia, accertata nel 2012, è stato attivato nella zona di ritrovamento un programma di monitoraggio teso a studiare l’insetto e il suo comportamento nell’ambiente. La specie desta maggiori preoccupazioni nel settore frutticolo, dove il danno rappresentato dalle tipiche deformazioni può giungere al 100%

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.
abstract

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
abstract

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

2015 - Preoccupano le infestazioni della cimice asiatica [Articolo su rivista]
Bortolotti, Paolo; Caruso, Stefano; Nannini, Roberta; Vaccari, Giacomo; Casoli, Luca; Bariselli, Massimo; Bortolini, Sara; Costi, Elena; Guidetti, Roberto; Maistrello, Lara
abstract

2015-Anno caldo delle cimici. Nel corso dell’estate le infestazioni di Halyomorpha halys hanno investito un territorio sempre più ampio, con gravi percentuali di danno su numerose colture. A esse si sono sovrapposti gli attacchi di altri pentatomidi, accomunati anch’essi dalla caratteristica di pungere e deprezzare i frutti. Il malessere degli agricoltori è crescente e la situazione in alcuni contesti ha assunto le dimensioni di un’autentica emergenza.

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
abstract

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
abstract

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.

2015 - Tardigrades of Sweden; an updated check-list [Articolo su rivista]
Guidetti, Roberto; Jönsson, K. Ingemar; Kristensen, M. Reinhardt
abstract

Tardigrades occur worldwide and in a variety of ecosystems and habitats representing an important component of the micrometazoan biodiversity. Several studies documenting the occurrence of tardigrades in Sweden have been published since the first reports in early 1900, but no comprehensive summary of these studies have been published. We compiled the available information on recorded tardigrades from Sweden, using material from published studies and museum and university collections. In total, our review document 101 species of tardigrades that have been recorded from Sweden (an updated checklist of tardigrades from Sweden will be available online), of which 14 species are new records for the country. The highest number of species was recorded in the northernmost province of Lappland and the more southern provinces of Uppland and Skåne, while much lower species numbers are reported from the middle part of Sweden. This pattern probably represents biased sampling activities of biologists rather than real differences in biodiversity of tardigrades. In view of the few studies that have been made on tardigrade biodiversity in Sweden, the relatively high number of tardigrade species recorded, representing almost a tenth of the species recorded worldwide, indicates that many more species remain to be found. In this respect, more studies of the marine ecosystems along the Swedish west coast and the long Baltic Sea coastline would be of particular interest.

2014 - Aquatic tardigrades in the Great Smoky Mountains National Park, North Carolina and Tennessee, U.S.A., with the description of a new species of Thulinius (Tardigrada, Isohypsibiidae) [Articolo su rivista]
Bertolani, Roberto; PAUL J., Bartels; Guidetti, Roberto; Cesari, Michele; DIANE R., Nelson
abstract

As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), an extensive survey of tardigrades has been conducted in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, U.S.A., by Bartels and Nelson. Freshwater tardigrades include three species in the aquatic genus Thulinius (Eutardigrada, Isohypsibiidae). A new species, Thulinius romanoi, described from stream sediment, is distinguished from all other congeners by having a sculptured cuticle. In addition, the presence of Thulinius augusti (Murray, 1907) was verified by combined orphological and molecular analysis, and nine specimens of a third species, Thulinius cf. saltursus, were also found. Thulinius augusti is a new record for the United States. Thulinius saltursus (Schuster, Toftner & Grigarick, 1978) was previously recorded in California and Ohio, but our specimens vary slightly in morphology. The list of tardigrades from streams in the GSMNP was updated to a total of 44 species, 22 of which were predominantly or exclusively aquatic.

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
abstract

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 - Cetacei del Museo di Zoologia e Anatomia Comparata dell’Università degli Studi di Modena e Reggio Emilia [Articolo su rivista]
Bisanti, Matteo; Pederzoli, Aurora; Guidetti, Roberto; Gambarelli, Andrea
abstract

Il Museo di Zoologia e Anatomia Comparata (MZAC) dell’Università degli Studi di Modena e Reggio Emilia ha una vasta collezione storica di vertebrati. Tra questi, pur essendo numericamente pochi, i reperti di cetacei sembrano avere un importante valore storico. Nel presente articolo viene presentato l’elenco dei reperti storici del MZAC e segnalate le nuove recenti acquisizioni.

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
abstract

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 - Halyomorpha halys in Italy: first results of field monitoring in fruit orchards [Abstract in Atti di Convegno]
Maistrello, Lara; Costi, Elena; Caruso, S.; Vaccari, Giacomo; Bortolotti, P.; Nannini, R.; Casoli, L.; Montermini, A.; Guidetti, Roberto
abstract

The invasive pest Brown Marmorated Stink Bug (BMSB) Halyomorpha halys (Heteroptera, Pentatomidae) was detected for the first time in Italy in September 2012 in Modena province (Northern Italy) during an insect collection for educational purposes. A survey performed in 2013 allowed to detect its presence in Emilia Romagna, Lombardy and Piedmont regions. In 2014, in the provinces of Modena, Reggio Emilia and Bologna a periodical active field monitoring was performed using tree beating, sweep-net and visual observations in selected orchards and vineyards, recording numbers of BMSB adults and nymphs, and of other Heteroptera. Besides, fruit injury and crop loss were recorded at harvest. Partial results from field data obtained between April and July 2014 are presented, indicating that BMSB is already becoming an important pest of fruit orchards and that special attention should be deserved to monitor its spread all over the region and the whole Italian country.

2014 - L’invasione della nuova specie aliena Halyomorpha halys (Heteroptera, Pentatomidae): diversità genetica e possibili aree d’originedelle popolazioni individuate in Italia e Canton Ticino [Abstract in Atti di Convegno]
Guidetti, Roberto; Cesari, Michele; Ganzerli, F.; Dioli, P.; Maistrello, Lara
abstract

La cimice bruna marmorizzata Halyomorpha halys è un pentatomide originario dell’Estremo Oriente. Negli ultimi anni si è diffusa nell’America Settentrionale (Canada e U.S.A.), causando ingenti danni economici alle colture. A partire dal 2007 H. halys è stata rilevata in Europa centrale (Svizzera, Francia e Germania) e, dal 2012, è stata segnalata anche in Italia. Da indagini effettuate nel 2013 è emerso che è presente con numeri anche consistenti di individui in Emilia (area modenese e province confinanti), Lombardia, Canton Ticino e Piemonte. Non essendo note le modalità di introduzione della cimice, per determinare il centro di origine degli esemplari rinvenuti in questo territorio, il presente studio ha analizzato una porzione del gene mitocondriale codificante per la citocromo c ossidasi sub unità I (cox1), di solito impiegata come codice a barre genetico per l’identificazione di specie (DNA barcoding) e l’ha impiegata quale marcatore molecolare per l’identificazione e studio della diversità genetica di H. halys. L’analisi di oltre 35 esemplari provenienti dall’Italia (Lombardia e Emilia-Romagna) e Svizzera (Canton Ticino) ha portato all’identificazione di due soli aplotipi, distinti tra loro per una sostituzione. Tutti gli esemplari lombardi e svizzeri presentavano il medesimo aplotipo, già ritrovato in letteratura anche in individui cinesi e svizzeri provenienti da altri cantoni elvetici. Il secondo aplotipo, finora mai ritrovato in Europa, è invece risultato comune per tutti gli esemplari provenienti dall’Emilia ed è condiviso con altri esemplari descritti in letteratura, originari sia dalla Cina che dal Nord America (Canada e U.S.A.). I dati attuali mostrano perciò come gli esemplari di H. halys presenti in Italia costituiscano almeno due diverse popolazioni che si sono originate da due distinti eventi di invasione. Probabilmente tali eventi sono stati causati dalla movimentazione di merci e persone. La popolazione lombarda potrebbe essersi originata a causa del trasporto passivo di esemplari dalla Svizzera o a causa della diffusione progressiva della popolazione svizzera attraverso la valle del Ticino. La popolazione emiliana invece potrebbe essersi originata a seguito del trasporto accidentale di individui attraverso scambi commerciali con il continente americano oppure con quello asiatico, dalla stessa regione da cui si è originata la popolazione nordamericana.

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
abstract

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
abstract

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 - Sulle tracce della biodiversità vegetale per riscoprire la biodiversità animale: esempio di uno studio integrato presso il Museo di Zoologia ed Anatomia Comparata di Modena [Articolo su rivista]
Pederzoli, Aurora; Mingucci, Marta; Mazzanti, Marta; Bosi, Giovanna; Guidetti, Roberto
abstract

In the Museum of Zoology and Comparative Anatomy of the Modena and Reggio Emilia University restoration works on mounted skin specimens have been recently done. Among primates, a triptych formed by a male, a female and a young specimens of Colobus guereza without the historical identification tag has been renewal. During the cleaning steps of the Guereza specimens fur, two mericarps belonging to Cynoglossum amplifolium were found. The distribution area of this species allowed to possibly identify the place of origin of Guereza specimens as Ethiopia.

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
abstract

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
abstract

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
abstract

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
abstract

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
abstract

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
abstract

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
abstract

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 - 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
abstract

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
abstract

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 - High level of phenotypic homoplasy amongst eutardigrades (Tardigrada) based on morphological and total evidence phylogenetic analyses [Articolo su rivista]
N., Guil; A., Machordom; Guidetti, Roberto
abstract

Much of what is known about the phylogenetic relationships of the neglected phylum Tardigrada comes from molecular data, rather than from morphology-based phylogenetic studies. Several molecular phylogenies have been proposed, but morphological and total evidence phylogenetic approaches are scarce. We performed the first morphological phylogeny (based on maximum parsimony and Bayesian inference) including all genera from the class Eutardigrada. Furthermore, we carried out a total evidence approach adding molecular information available in public databases. We compared the morphological and total evidence phylogenies with current molecular hypotheses and Eutardigrada classifications, which our results partially support. These classifications were supported only when homoplastic characters (related to the buccopharyngeal apparatus) were excluded. The importance of morphological phylogenies is discussed as well as their utility for questioning current phylogenetic hypotheses and classifications based solely on molecular information. Lastly, we propose evolutionary hypotheses about eutardigrade phylogenetic relationships that should be tested based on our morphological results.

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
abstract

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
abstract

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

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
abstract

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.
abstract

Tardigrades: multicellular organisms aboard the International Space Station

2013 - The morphological and molecular analyses of a new South American urban tardigrade offer new insights on the biological meaning of theMacrobiotus hufelandigroup of species (Tardigrada: Macrobiotidae) [Articolo su rivista]
Guidetti, Roberto; Julio Ricardo, Peluffo; Alejandra Mariana, Rocha; Cesari, Michele; María Cristina Moly de, Peluffo
abstract

Worldwide knowledge of tardigrade fauna is still limited, and many areas such as South America are not well studied. The collection of new substrates in Argentinean urban areas provided an opportunity to describe the new tardigrade species Macrobiotus kristenseni sp. nov. This species has been studied with an integrative taxonomic approach, analysing its morphology by light and scanning electron microscopy, and considering two genes (cox1 and 18S rRNA) for DNA barcoding and phylogenetic purposes. The species belongs to the Macrobiotus hufelandi group of species, and it is characterized by egg processes in the shape of elongated cones with truncated and enlarged apexes, and by a high genetic distance with respect to closely related species (cox1: 18.2–33.4%).Morphological and molecular data show that the Macrobiotus hufelandi group of species has to be considered a true biological entity; one of the more widespread tardigrade lineages in continental environments.

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
abstract

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
abstract

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
abstract

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
abstract

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.
abstract

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 - 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
abstract

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

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 - 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
abstract

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

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

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
abstract

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 - Sulle orme di Darwin: alla ricerca del progenitore comune [Capitolo/Saggio]
Guidetti, Roberto
abstract

Approcci filosofici e metodologici per derivare e rappresentare le relazioni evolutive tra gli organismi

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
abstract

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
abstract

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
abstract

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

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 - Phylum Tardigrada Doyère, 1840 [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto
abstract

Updated taxonomy of the phylum Tardigrada

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
abstract

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
abstract

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
abstract

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
abstract

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

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

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

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

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 - I visitatori hanno detto di noi. [Articolo su rivista]
Guidetti, Roberto; Bertacchini, Milena
abstract

Indagine sui 30000 visitatori della mostra su Darwin realizzata a Modena nel 2009-2010.

2010 - La collezione erpetologica del Museo di Zoologia ed Anatomia Comparata dell’Università di Modena e Reggio Emilia [Articolo su rivista]
G., Viotti; Pederzoli, Aurora; Guidetti, Roberto
abstract

Il Museo di Zoologia e Anatomia Comparata dell’Università di Modena e Reggio Emilia ospita diverse collezioni zoologiche prevalentemente risalenti ai secoli XIX e XX. Tali collezioni necessitano di interventi di restauro, di revisioni tassonomiche e di una catalogazione aggiornata.La collezione erpetologica risulta essere composta da 309 esemplari appartenenti a 149 specie. In particolare gli anfibi sono rappresentati da 61 esemplari, di cui 10 appartenenti all’ordine Caudata e i restanti 51 appartenenti all’ordine Anura, per un totale di 37 specie. Gli esemplari di anfibi provengono dal territorio modenese, altri provengono dall’Italia meridionale ma molti provengono dal Sudamerica, oltre ad alcune specie di origine africana. Per quanto riguarda i rettili, il museo conta 248 esemplari, di cui 210 appartenenti all’ordine Squamata (106 Ofidi, 104 Sauri), 27 appartenenti all’ordine Testudines e 11 appartenenti all’ordine Crocodylia. La provenienza è estremamente varia, accanto a numerosi esemplari italiani ne troviamo moltissimi provenienti da Asia, Africa, Americhe e Australia.

2010 - Progetto integrato sull'evoluzione come concetto unificante delle Scienze Naturali: tematiche diverse ma interagenti. [Relazione in Atti di Convegno]
Fioroni, Chiara; Fregni, Paola; Guidetti, Roberto; Pederzoli, Aurora; Pini, Lorenza; Veronesi, Paola
abstract

-Numerosi sono gli argomenti che si possono prestare all’integrazione fra discipline scientifiche e in special modoquelle afferenti alle scienze della natura che risultano imprescindibili l’una dall’altra.Poiché l’evoluzione fornisce importanti nozioni e spiega i meccanismi fondamentali per la comprensione delmondo naturale, i curricula didattici e gli insegnanti dovrebbero enfatizzare la sua importanza come concettounificante delle Scienze Naturali per la poliedricità di argomenti coinvolti.Vista la rilevanza della tematica si è sentita la necessità di elaborare una proposta didattica che costituisse unapossibile risposta al problema presentato.

2010 - Sulle tracce della biodiversità vegetale per riscoprire la biodiversità animale: uno studio integrato presso il Museo di Zoologia [Abstract in Rivista]
Pederzoli, Aurora; M., Mingucci; Mazzanti, Marta; Bosi, Giovanna; Guidetti, Roberto
abstract

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2010 - Tardigrada [Capitolo/Saggio]
D., Nelson; Guidetti, Roberto; Rebecchi, Lorena
abstract

.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
abstract

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 - L'evoluzione come concetto unificante delle Scienze Naturali [Abstract in Atti di Convegno]
Fioroni, Chiara; Fregni, Paola; Guidetti, Roberto; Pederzoli, Aurora; Pini, L.; Veronesi, P.
abstract

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2009 - New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov. [Articolo su rivista]
Guidetti, Roberto; R. O., Schill; Bertolani, Roberto; T., Dandekar; M., Wolf
abstract

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus, corresponding to the richtersi-areolatus group. Moreover, cryptic species have been identified within the Macrobiotus richtersi group and within Richtersius. Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the richtersi-areolatus group.

2009 - Osservare gli animali per capire l'evoluzione: scelte tematiche ed espositive all'interno della mostra "Darwin: Modena e 200 anni di evoluzione" [Articolo su rivista]
Guidetti, Roberto; Pederzoli, Aurora
abstract

Vengono discusse le tematiche ed espositive effettuate nella trattazione di tematiche biologiche e evolutive all'interno della mostra "Darwin: Modena e 200 anni di evoluzione"

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
abstract

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
abstract

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
abstract

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.

2009 - Una mostra dedicata a Charles Darwin e le meraviglie delle scienze naturali [Articolo su rivista]
Bertacchini, Milena; Guidetti, Roberto
abstract

Vengono trattate le ragioni che hanno indotto l'allestimento di una esposizione temporanea su Darwin a Modena e le riflessioni scientifiche e culturali che hanno guidato la scelta dei contenuti

2009 - Yours faithfully and obliged Charles Darwin: la lettera di Darwin alla Società dei Naturalisti in Modena [Articolo su rivista]
Guidetti, Roberto
abstract

Vengono raccontate le vicissitudini del ritrovamento di una lettera autografa di Darwin alla Società dei Naturalisti in Modena. Viene valutata la sua autenticità e se ne ripercorre la storia.

2008 - Cryptic Species in Macrobiotus (Tardigrada): A Morphological and Molecular Approach [Abstract in Atti di Convegno]
Guidetti, Roberto; Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele
abstract

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
abstract

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
abstract

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 - La collezione entomologica del Museo di Zoologia ed Anatomia Comparata dell’Università di Modena e Reggio Emilia [Articolo su rivista]
Bisanti, Matteo; Pederzoli, Aurora; Guidetti, Roberto
abstract

Negli ultimi anni è nata l'esigenza di creare un database completo delle varie collezioni presenti all'interno del Museo di Zoologia ed Anatomia Comparata dell?università di Modena e Reggio Emilia in modo da poterne quantificare il reale valore scientifico e storico. Questa esigenza trova risposta oggi nel catalogo della collezione entomologica museale qui propostoche, oltre a puntualizzare lo stato attuale della collezione, si propone anche come base dinamica, suscettibile di aggiornamento e miglioramento, per successive ricerche scientifiche e di carattere storico.

2008 - Museo di Zoologia e Anatomia Comparata [Capitolo/Saggio]
Ansaloni, Ivano; Pederzoli, Aurora; Guidetti, Roberto; Baraldi, Laura
abstract

Vengono trattati diversi aspetti del Museo di Zoologia e Anatomia comaparata dell'Universotà di Modena: storia, attività dei conservatori, direttori, acquisizioni;attività didattica

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
abstract

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
abstract

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
abstract

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

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

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
abstract

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
abstract

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 - Fantastic animals as an experimental model to teach animal adaptation [Articolo su rivista]
Guidetti, Roberto; L., Baraldi; C., Calzolari; L., Pini; P., Veronesi; Pederzoli, Aurora
abstract

Background: Science curricula and teachers should emphasize evolution in a mannercommensurate with its importance as a unifying concept in science. The concept of adaptationrepresents a first step to understand the results of natural selection. We settled an experimentalproject of alternative didactic to improve knowledge of organism adaptation. Students wereinvolved and stimulated in learning processes by creative activities. To set adaptation in a historicframe, fossil records as evidence of past life and evolution were considered.Results: The experimental project is schematized in nine phases: review of previous knowledge;lesson on fossils; lesson on fantastic animals; planning an imaginary world; creation of an imaginaryanimal; revision of the imaginary animals; adaptations of real animals; adaptations of fossil animals;and public exposition. A rubric to evaluate the student's performances is reported. The projectinvolved professors and students of the University of Modena and Reggio Emilia and of the "G.Marconi" Secondary School of First Degree (Modena, Italy).Conclusion: The educational objectives of the project are in line with the National Indications ofthe Italian Ministry of Public Instruction: knowledge of the characteristics of living beings, themeanings of the term "adaptation", the meaning of fossils, the definition of ecosystem, and theparticularity of the different biomes. At the end of the project, students will be able to graspparticular adaptations of real organisms and to deduce information about the environment in whichthe organism evolved. This project allows students to review previous knowledge and to form theirpersonalities.

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
abstract

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 - New taxonomic position of several Macrobiotus species (Eutardigrada: Macrobiotidae) [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto; P., Degma
abstract

The main character distinguishing Minibiotus from Macrobiotus is the presence of peribuccal papulae instead of lamellae.However, most Minibiotus species also share a set of characters of the bucco-pharyngeal apparatus. This set isdefined by ten morphological features (the first seven common to all species, the last three to most of them): antero-ventralmouth (1), teeth in oral cavity absent or strongly reduced (2), a rigid and narrow buccal tube (3), buccal tube wallthickened below the point of insertion of stylet supports (4), stylet supports inserted at considerable distance from theposterior end of the buccal tube (5), short ventral lamina (6), short macroplacoid row length (7), a double curvature of thebuccal tube (8), first macroplacoid situated very close to the pharyngeal apophysis (9) and three almost rounded macroplacoids(10). We examined the original descriptions and/or the type material of several species belonging to the genusMacrobiotus that seemed to present this set of characters. It was concluded that several species should be transferredfrom Macrobiotus to Minibiotus. The following new combinations are proposed: Minibiotus subintermedius (Ramazzotti,1962) comb. n., Minibiotus pustulatus (Ramazzotti, 1959) comb. n., Minibiotus julietae (de Barros, 1942) comb.n., Minibiotus marcusi (de Barros, 1942) comb. n., Minibiotus granatai (Pardi, 1941) comb. n., Minibiotus allani (Murray,1913) comb. n., Minibiotus crassidens (Murray, 1907) comb. n. and Minibiotus acontistus (de Barros, 1942) comb.n.. As a result of these new combinations, the genera Minibiotus and Macrobiotus appear to be more homogeneous, buttheir monophyly has not yet been demonstrated. Further data are necessary to better analyze the systematic position ofMacrobiotus lazzaroi Maucci, 1986, Macrobiotus spertii Ramazzotti, 1957 and Macrobiotus striatus Mihelčič, 1949.

2007 - Notes to the current checklist of Tardigrada [Articolo su rivista]
Degma, P; Guidetti, Roberto
abstract

Corrections and additions to the recently published checklist of Tardigrada taxa (Guidetti & Bertolani 2005) are defined and described until the acceptance of this article. Two new synonyms are stated here: Hypsibius iharosi Bartoš, 1941 syn. nov. and Isohypsibius gibbus (Marcus, 1928) syn. nov.

2007 - Observations on the "tenuis group" (Eutardigrada, Macrobiotidae) and description of a new Macrobiotus species [Articolo su rivista]
Guil, N; Guidetti, Roberto; A., Machordom
abstract

A new species of the tardigrade genus Macrobiotus is described. The species, designated M. ciprianoi n.sp., was isolated from a mixture of Provence broom leaf litter and mosses, and from rock mossescollected in the Sierra de Guadarrama, Madrid (Spain). Given that Macrobiotus ciprianoi n. sp. sharesseveral characters to members of the ‘‘tenuis group’’, we assessed the taxonomic homogeneity of thegroup. The new species differs from those of the ‘‘tenuis group’’ according to a unique set ofcharacters related with claw shape, features of the buccal-pharyngeal apparatus, and egg morphology.Our analysis of holotypes and/or paratypes of ‘‘tenuis group’’ species and other Macrobiotus specieswith similar characters (M. bondavallii and M. caelicola) reflects the heterogeneity of this group ofspecies as currently described.

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
abstract

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

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

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

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

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
abstract

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.

2006 - DESICCATION TOLERANCE AND HEAT-SHOCK PROTEINS EXPRESSION IN ACTIVE AND DORMANT AMPHIBOLUS VOLUBILUS (EUTARDIGRADA) [Abstract in Atti di Convegno]
Boschini, Deborah; Guidetti, Roberto; Rebecchi, Lorena
abstract

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 - Dal passato....al futuro: prove di adattamento [Abstract in Atti di Convegno]
Guidetti, Roberto; C., Calzolari; L., Baraldi; L., Pini; P., Veronesi; Pederzoli, Aurora
abstract

L'attività presentata è un'esperienza di laboratorio scientifico riguardante gli organismi fossili e il concetto di adattamento portata avanti da un gruppo di alunni di terza media.L'attività è nata come progetto di didattica alternativa per motivare gli studenti all'acquisizione di nuove conoscenze.

2006 - Description of the new species Calcarobiotus (C.) longinoi sp nov (Eutardigrada, Macrobiotidae) from Costa Rica with the diagnostic key to the genus Calcarobiotus [Articolo su rivista]
L., Kaczmarek; L., Michalczyk; Guidetti, Roberto
abstract

A new species of Tardigrada (Eutardigrada), Calcarobiotus longinoi sp. nov., is described from moss samples collected in Alajuela Province (Costa Rica). The presence of genus Calcarobiotus is new for Central America. Ibis species differs from other species of the genus Calcarobiotus by lacking of spurs on the basal portion of the claws. On the base of this new finding, the diagnosis of the subgenus Calcarobiotus is emended. A taxonomic key to the genus Calcarobiotus is also presented.

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

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
abstract

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
abstract

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
abstract

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).

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
abstract

I tardigradi rappresentano un modello animale particolarmente valido per la comprensione dei meccanismi, a livello organismico e cellulare, di protezione dagli stress dell’ambiente spaziale. Sono infatti invertebrati microscopici (circa 0,3-0,5 mm) che per condurre vita attiva necessitano di almeno un velo d’acqua, ma quando questa non è disponibile, rallentano notevolmente, o sospendono, il metabolismo sopravvivendo essiccati (anidrobiosi), o congelati (criobiosi). Entrando in anidrobiosi producono bioprotettori (es. trealosio) che stabilizzano le membrane cellulari. In anidrobiosi resistono inoltre ad ulteriori stress, come ad esempio, temperature di 150°C, o vicine allo zero assoluto (-273°C), radiazioni ionizzanti 500 volte superiori a quelle che porterebbero all’immediata morte dell’uomo, atmosfere sature di CO2 e HNO3, immersione in alcoli e pressioni di 600 Mpa (Bertolani et al., 2004). I tardigradi attuano la criptobiosi (anidrobiosi e criobiosi) in qualunque fase del ciclo vitale; alcune specie possono essere allevate (Altiero & Rebecchi, 2001) risultando particolarmente longeve, tanto da superare l’anno di vita (Altiero, Rebecchi & Bertolani, 2006). Inoltre, a differenza di altri minuscoli metazoi, sono caratterizzati da divisioni cellulari anche nell’adulto. Indagini dell’U.O. sulle capacità criptobiotiche dei tardigradi (parzialmente finanziate da ASI, 2001) hanno consentito di dimostrare la presenza di differenze intra- ed interspecifiche nella sopravvivenza in anidrobiosi (Jönsson, Borsari & Rebecchi, 2001), la relazione fra tratti fenotipici dei tardigradi e sopravvivenza all’essiccamento (Jönsson & Rebecchi, 2002), la sopravvivenza a lungo termine in anidrobiosi (Rebecchi et al., 2006), la presenza di differenze interspecifiche nella capacità di resistere al congelamento (Guidetti et al., inviato).L’ambiente spaziale provoca sui viventi stress che accelerano il tasso di invecchiamento; per individuare strategie contro l’invecchiamento ed anche al fine di applicazioni biotecnologiche, viene qui proposto lo studio dei meccanismi molecolari che consentono ai tardigradi di resistere agli stress tipici di tale ambiente, in modo da acquisire conoscenze sui meccanismi di riparazione e mantenimento di cellule e organismi. In questa prospettiva si colloca dunque il progetto ASSC-TARSE (TArdigrade Resistance to Space Effect), inserito nell’area II (Cellule, Tessuti, Organi) del progetto MoMa. Il progetto ASSC-TARSE, utilizzando tardigradi in toto e loro cellule (storage cells) in coltura primaria, si propone di: i. valutare la sopravvivenza dei tardigradi dopo stress quali essiccamento, microgravità e radiazioni attraverso il confronto fra animali attivi e anidrobionti; ii. acquisire informazioni sulla fisiologia, la biochimica e il genoma dei tardigradi a seguito di tali stress; iii. acquisire informazioni sull’apoptosi. Durante l’elaborazione del progetto, l’U.O. ha messo a punto i protocolli per l’allestimento di colture primarie di storage cells, l’analisi del DNA genomico e l’analisi dell’espressione di Heat-shock proteins (Hsp) nei tardigradi attivi ed anidrobionti. La disponibilità di tali protocolli, oltre all’ottenuta dimostrazione che i tardigradi essiccati esprimono più Hsp70 rispetto a quelli attivi, rappresentano una valida base di partenza per il raggiungimento degli obiettivi sopraindicati. La piena realizzazione del progetto TARSE consentirà di ottenere i seguenti risultati: i. la rilevazione degli effetti dell’essiccamento, delle radiazioni e della microgravità sulla sopravvivenza sia di organismi in toto che di cellule in coltura; ii. la rilevazione di probabili danni al DNA genomico e dell’eventuale capacità di riparo; iii. la rilevazione dei fenomeni di apoptosi; iv. l’individuazione e la quantificazione di bioprotettori coinvolti nei meccanismi di resistenza all’essiccamento e alle radiazioni: Heat-sho

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

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

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

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.
abstract

2005 - A new species of Tardigrada (Eutardigrada : Macrobiotidae) from Iberian peninsula and Canary Islands (Spain) [Articolo su rivista]
Guil, N; Guidetti, Roberto
abstract

Minibiotus gumersindoi n. sp. is described. This species was collected on a granite mountain range in Sierra de Guadarrama ( Bustarviejo valley, Embalse de la Jarosa and Cercedilla, Madrid, Spain) and on a volcanic island in Parque Natural Caldera de Taburiente ( La Palma, Canary Islands, Spain). This new species is characterized by a unique set of characters in the genus: the presence of large round pores in the body and a larger round pore at the distal part of each leg. New records of several known species, for Madrid, Canary Islands and/or the Iberian Peninsula are given.

2005 - Al museo della Bilancia per scoprire giocando gli adattamenti animali alla forza di gravità [Abstract in Atti di Convegno]
Pederzoli, Aurora; L., Baraldi; D., Dell'Orco; G., Luppi; M., Nicolini; Guidetti, Roberto
abstract

In occasione di una Mostra sulla forza di gravità allestita presso il Museo della Bilancia di Campogalliano (MO) sono state organizzate postazioni interattive per sperimentare la forza di gravità. In particolare con "crescere con la gravità" sono stati mostrati gli adattamenti sviluppati da alcuni animali in relazione a questa forza.

2005 - Phylogenetic analysis of Macrobiotidae (Eutardigrada, Parachela): a combined morphological and molecular approach [Articolo su rivista]
Guidetti, Roberto; A., Gandolfi; V., Rossi; Bertolani, Roberto
abstract

Combined analyses of morphological and molecular data were used to resolve phylogenetic relationships within Macrobiotidae (Eutardigrada). Morphological data were analysed using a cladistic approach with a matrix comprising 15 taxa with 17 characters to obtain a phylogenetic reconstruction. Molecular data were obtained by sequencing the cytochrome c oxidase subunit I gene in seven species of Macrobiotidae and one of Eohypsibiidae (used as outgroup). The morphological character defining the family, symmetrical claw on each leg, turns out to be plesiomorphic. Moreover, neither morphological nor molecular analyses supports a monophyletic clade for the subfamily Macrobiotinae, whereas both support a well defined evolutionary line (Murrayinae) within the family. We propose elevating the latter to family level (Murrayidae) while temporarily retaining as valid the family Macrobiotidae (amending its diagnosis, including within it only Macrobiotinae). Murrayidae opens an interesting evolutionary prospective, because the entire line has differentiated without sexual reproduction, constituting an example of evolution of asexual lineages.

2005 - Sfidando la gravità con l'uomo ragno....strategie comunicative per argomenti difficili! [Abstract in Atti di Convegno]
L., Baraldi; Pederzoli, Aurora; Guidetti, Roberto
abstract

E'stato realizzato un percorso didattico multidisciplinare su CD-ROM con il supporto scientifico del Dipartimento di Biologia animale e del Dipartimento di Fisica dell'Università di Modena e Reggio Emilia da parte di specializzandi della SSIS (Scuola di Specializzazione per l'Insegnamento Secondario avente per tema gli adattamenti alla gravità.

2005 - Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto
abstract

Since the most recent monograph on Tardigrada by Ramazzotti & Maucci (1983), many changes have been introduced in the systematics of this phylum which has gone through different revisions and has been expanded by several new taxa. Moreover, the number of species is considerably increased (almost doubled to more than 960 species) and new families and genera have been created, while many taxa have been amended. The difficulty in finding a complete, updated list of taxa and the problems with putting together all the newly proposed taxonomic criteria, led us to compile an updated list of tardigrade taxa. This list is supplemented by a series of bibliographic references inherent in the synonymies and/or the amended diagnosis of the taxa occurred after 1983, and a list of the main characters used in the diagnosis of the species. These lists are aimed at helping not only the specialists studying this group, but also researchers interested in biodiversity and those who have to refer to this animal group.

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

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 - L'uomo in cima alla scala............rischia di cadere: proposte didattiche per l'insegnamento dell'evoluzione nelle Scuole Secondarie di Primo grado. [Abstract in Atti di Convegno]
Guidetti, Roberto; Pederzoli, Aurora; S., Barbieri; M. A., Pinotti; L., Uccellari; A., Uccelli
abstract

Nell'ambito del corso di Scienze della vita con laboratorio didattico della SSIS ( Scuola di Specializzazione per l'Insegnamento Secondario) di Modena è stato discusso il concetto di evoluzione e prodotta una serie di Unità Didattiche per la Scuola Secondaria di Primo Grado.

2004 - Studies on groundwater tardigrades of Lessinian Massif (Northern Italy). [Abstract in Atti di Convegno]
Guidetti, Roberto; D. M. P., Galassi; Bertolani, Roberto
abstract

Terrestrial tardigrades require a film of water around the body to conduct an active life. Therefore, in several cases the distinction between freshwater and terrestrial species is unclear as some species can tolerate a wide range of moisture regimes. Less than 6% of species are considered to inhabit permanent freshwater habitats. These species live as epiphyte on aquatic plants, benthic algal mats or among the minute voids of sediments in lotic and lentic waters. Tardigrades are sporadically reported from different groundwater habitats but little data are still available on their ecological affinity for this environment, as well as on their distribution and diversity in groundwater at different spatial scales. A stratified random sampling in both karstic and porous aquifers in the Lessinian Massif (northern Italy) gave the opportunity to asses their distribution patterns on a regional scale and to better define some ecological features of the collected species.Eight tardigrade species were collected in 29 of 129 sampling sites. These sites are located in five hydrogeographic basins in the selected region. Tardigrades were distributed in almost all the groundwater habitats; in saturated porous (wells), in saturated karst (only spring habitats) and in these cases always linked to the presence of unconsolidated sediments, and especially in hyporheic habitats, where they appeared with the highest frequency of occurrence and the highest abundance. They belong to six genera and two families of eutardigrades. Four species belong to typical limnic genera (Pseudobiotus, Thulinius and Dactylobiotus) and are characterized by claws longer than those of moss or soil-dwelling species. One species of Diphascon was already reported from groundwater, while two species of Macrobiotus and one of Hypsibius are easily found in terrestrial habitats. Some species of tardigrades collected in surface hyporheic habitats possess eye-spots. From an ecological point of view, most of them may be considered stygoxene or possibly stygophilous species. The morphological features of these species are similar to those of species living in surface freshwater on in the leaf-litter. This observation highlights the potential role of several habitats located at the interface surface/subsurface environments as potential migration ways for several species to enter groundwater. For the same reason, such a physical continuum and some degree of convergence in habitat features may determine the absence of clear morphological distinction between species living in such interface and those living in true groundwater.This research is partially granted by the EU-Pascalis project and by 60% MIUR from the University of Modena and Reggio Emilia.

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
abstract

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
abstract

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 - Revision of the genus Pseudodiphascon (Tardigrada, Macrobiotidae), with the erection of three new genera [Articolo su rivista]
Guidetti, Roberto; Pilato, G.
abstract

A flexible but non-annulated buccal tube was used to erect the subgenus Pseudodiphascon , later raised to genus level (type species Macrobiotus inflexus ). However, the presence of a flexible (but non-annulated) buccal tube in the holotype and paratype is due to the moulting simplex stage of both animals. Therefore Macrobiotus inflexus must be considered species dubia . The other species to date attributed to Pseudodiphascon differ from the type species of the genus in the presence of a pharyngeal tube that appears annulated. Because of this and other characters, they do not conform to the definition of Pseudodiphascon and cannot be attributed to this genus, which must be considered genus dubium . The characters of the claws and of the buccal-pharyngeal apparatuses led us to describe three new genera: Biserovus gen. n.: claws without a differentiated basal portion, annulated pharyngeal tube, peribuccal lamellae present; Minilentus gen. n.: claws of ' hufelandi type', annulated pharyngeal tube, peribuccal lamellae absent; Insuetifurca gen. n.: claws of modified ' hufelandi type', annulated pharyngeal tube, peribuccal lamellae present, stylet furcae of a peculiar shape. Pseudodiphascon diphasconides lacks annulation in the buccal tube and is transferred to the genus Minibiotus .

2002 - Long-term anhydrobiotic survival in semi-terrestrial micrometazoans [Articolo su rivista]
Guidetti, Roberto; K. I., Jonsson
abstract

This study represents the first systematic investigation of long-term anhydrobiotic survival in tardigrades, rotifers and nematodes inhabiting mosses and lichens. Sixty-three different samples from public and private collections, kept dry for 9-138 years, were examined. Rotifers of the genus Mniohia and the eutardigrade Ramazzottius oberhaeuseri (hatched from eggs) were found alive from one of the samples (9 years old). These observations represent the longest record for rotifers in the anhydrobiotic state. For tardigrades, our results confirm previous reports on the upper limit of anhydrobiotic survival under atmospheric oxygen conditions. This study suggests the possibility that tardigrade eggs are able to withstand longer periods in anhydrobiosis than animals. Some problems related to the evaluation of long-term anhydrobiotic survival, such as contamination and chemical treatments of samples, are reported. The possible role of the microenvironment in which the anhydrobiotic animals are kept is discussed.

2001 - An evolutionary line of the Macrobiotinae (Tardigrada, Macrobiotidae): Calcarobiotus and related species [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto
abstract

Three species of Macrobiotus (M. gildae, M. polygonatus and M. eugranulatus) were re-examined. Based on their synapomorphies with Calcarobiotus, they are ascribed to that genus. Within Calcarobiouts two phyletic lines are detected on the basis of the claw morphology. Therefore a new subgenus is erected: Calcarobiotus (Discrepunguis) subgen. n. constituted by C. (D.) polygonatus comb. n., and C. (D.) eugranulatus comb. n.; the subgenus Calcarobiotus (Calcarobiotus) includes C (C) filmeri, C. (C) occultus, C. (C.) imperialis and C. (C.) gildae comb. n.

2001 - Effects of methyl bromide fumigation on anhydrobiotic micrometazoans [Articolo su rivista]
Jonsson, K. I.; Guidetti, Roberto
abstract

Anhydrobiotic organisms are well known for their resistance to extreme environmental conditions while in the inactive, ametabolic, state. Our study confirms that this resistance also transfers to treatments with the fumigant methyl bromide. We found alive specimens of nematodes, rotifers and tardigrades after 70 h treatment with methyl bromide. Quantitative estimates of survival in the eutardigrade Richtersius coronifer revealed no difference between treated and untreated samples which had been kept dry for similar times. However, R. coronifer from samples collected 11 months before treatment had lower survival compared to samples collected shortly before the treatment. The fact that anhydrobiotic organisms escape treatments with biocides should be considered a serious problem, both for the food industry and from the perspective of human mediated spread of organisms.

2001 - Phylogenetic relationships in the Macrobiotidae (Tardigrada : Eutardigrada : Parachela) [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto
abstract

Characters of the claws, buccal-pharyngeal apparatus and ultrastructure of the cuticle have been examined to identify phyletic relationships within the Macrobiotidae. Several apomorphies have been identified. The resulting cladogram shows two main phyletic lines within the family, corresponding to the subfamilies Macrobiotinae and Murrayinae. The relationships among the genera of the Murrayinae are resolved, whereas in the Macro biotinae five main phyletic lines are identified, whose relationships are still unknown. Pseudodiphascon and Macrobiotus appear to be polyphyletic genera.

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
abstract

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 Tardigrades of Emilia (Italy). III. Piane di Mocogno (Northern Apennines). [Articolo su rivista]
Guidetti, Roberto; Bertolani, Roberto
abstract

Samples of leaf litter, mosses and lichens were collected in a beech forest at Piane di Mocogno (Emilia, Italy), 1200 m asl. The tardigrade community of the leaf litter was similar to that found in this substrate in Italy and in the U.S.A. At Piane di Mocogno the species association within the leaf litter was different from that in mosses and lichens (which have similar fauna), confirming that very different communities characterise different substrates such as leaf litter and mosses. During this faunal analysis, six species of tardigrades were found for the first time in this area. The presence of Microhypsibius bertolanii (first record in leaf litter and in Italy) and Eohypsibius nadjae are note worthy. Ornamented eggs in an exuvium of Hypsibius cf. scabropygus were also recorded for the first time.

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

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.

1999 - Ecological and faunistic studies on tardigrades in leaf litter of beach forests [Articolo su rivista]
Guidetti, Roberto; Dr, Nelson; Bertolani, Roberto
abstract

Tardigrades in leaf litter were collected from beech forests on Roan Mountain (TN, U.S.A.) and Modenese Apennine (Italy) at different altitudes and during different seasons. Twenty-six species of eutardigrades and 6 species of heterotardigrades were found on Room Mountain and 20 species of eutardigrades on Apennine. In both cases the tardigrade communities showed great biodiversity and a similar species composition, represented by hygrophilous and eurytypic taxa. Differences in the number of specimens and the species composition were found, related to substrate layers, altitudes and seasons.

1998 - Two new species of Macrobiotidae (Tardigrada : Eutardigrada) from the United States of America, and some taxonomic considerations of the genus Murrayon [Articolo su rivista]
Guidetti, Roberto
abstract

Two new species of eutardigrades, Macrobiotus nelsonae and Murrayon stellatus, collected in Tennessee, U.S.A., are described. Macrobiotus hibernicus and Macrobiotus dianeae, related to M. stellatus, are re-examined and ascribed to the genus Murrayon on the basis of the claw type and the presence of a ventral hook on the strengthening bar. A dichotomous key to the genus Murrayon is proposed.

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

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

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

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