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Michele CESARI

Ricercatore t.d. art. 24 c. 3 lett. B
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 - 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 - 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 - 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 - Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. (Eutardigrada, Macrobiotidae, hufelandi group) [Articolo su rivista]
Ben Marnissi, Jamila; Cesari, Michele; Rebecchi, Lorena; Bertolani, Roberto
abstract

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 - 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 - 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 - 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.

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.

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 - 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.

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 - 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.

2016 - Ecological genetics of Italian peach-potato aphid (Myzus persicae) populations in relation to geography, dispersal and insecticide resistance as studied using microsatellite and resistance markers [Articolo su rivista]
Monti, Valentina; Loxdale, Hugh D.; Cesari, Michele; Frattini, Milo; Panini, Michela; Mazzoni, Emanuele; Manicardi, Gian Carlo
abstract

1 Eight polymorphic microsatellites and two insecticide resistance markers (kdr and R81T) were used to investigate the population genetics and demography of resistance mechanisms in field populations of the peach-potato aphid, Myzus persicae (Sulzer), a global crop pest, in several areas of Italy, notably the north-east, aiming to determine whether these parameters are linked to population structuring resulting from insecticide selective sweeps. 2 Individuals collected directly from peach orchards (main primary host) in the spring, especially after control failure, revealed 90 microsatellite alleles, of which approximately one-third were rare. 3 Many populations deviated from Hardy–Weinberg expectations, in a few instances as a result of null alleles. FIS values were on average negative (indicating heterozygote excess), whereas FST values (mean=0.137) reflected moderate interpopulation gene flow, with nonstructured populations having an estimated higher level than insecticide-treated ones. Population assignment and Mantel testing showed a non-homogenous pattern, with some populations isolated genetically. Linkage disequilibrium was detected in four of the microsatellites and both resistance markers. Contrasting FST patterns with resistance status supported the view that some populations were indeed structured. 4 Neighbour-joining trees revealed some populations to have similar insecticide resistance profiles, independent of geographical origin. 5 Overall, the findings reveal that the natural M. persicae populations investigated in the present study, living within an intensively cultivated agro-ecosystem, bear the signature of such management in terms of population structuring, as well as possibly also dynamics.

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 - 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 - 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 - 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 - 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.

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 - 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 - 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 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 - 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 - 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 impact of fire on terrestrial tardigrade biodiversity: a first case-study from Portugal [Articolo su rivista]
Filipe, Vicente; Cesari, Michele; Artur, Serrano; Bertolani, Roberto
abstract

Currently, loss of habitat is the greatest threat to biodiversity, yet little is known about its effect on microscopic animal taxa, such as Tardigrada. One of the causes of habitat destruction is forest fire, both natural and anthropogenic. The latter is commonly used in agriculture to kill insect pests, as a soil preparation, or conservation to create habitat mosaics. In Portugal, 42% of fire frequency is anthropogenic. There is no consensus on the impact of fires on biodiversity, with studies pointing towards different conclusions. Different methods and target taxonomic study groups may partly explain this paradigm. This study is the first into possible effects of habitat destruction on tardigrade populations, in which we analysed the taxonomic and genetic variations of tardigrades from a fire affected location in a Portuguese natural park.. Sampling was performed over 10-year period, from 2000 to 2010. The location was affected by a small fire in 1998 and a big fire in 2003. A total of 11 species from nine separate genera was recorded, from which 19 cox1 haplotypes were identified. Our data suggest a negative effect of a forest fire on tardigrade populations. Taxonomic and genetic richness, as well as abundance show lower levels in the years after a fire, compared with the preceding years. Additionally, the population recovered visibly faster after the small fire than after the bigger one. This is consistent with larger fires destroying larger forest areas, leaving fewer animals at a farther distance available for re-colonisation. Most species found before the main fire are also found after it, indicating these tardigrades have a high recolonisation capability. However, only three of all recorded haplotypes were found both pre and post the main fire, which indicates genetic diversity loss as a direct consequence of fire. Therefore, we conclude that habitat destruction by means of forest fire has a detrimental effect on tardigrade biodiversity, and may have similar effects on other small animals.

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.

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

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

2012 - A DNA barcoding approach in the study of tardigrades. [Abstract in Atti di Convegno]
Cesari, Michele; Rebecchi, Lorena; Guidetti, Roberto; Giovannini, Ilaria; Mori, L.; Bertolani, Roberto
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 - Identification of the trehalose-6-phosphate synthase (tps) gene in desiccation tolerant and intolerant tardigrades [Articolo su rivista]
Cesari, Michele; Altiero, Tiziana; Rebecchi, Lorena
abstract

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

2012 - L'approccio di DNA barcoding nello studio dei tardigradi [Abstract in Atti di Convegno]
Bertolani, Roberto; Rebecchi, Lorena; Guidetti, Roberto; Altiero, Tiziana; Giovannini, Ilaria; Mori, L.; Cesari, Michele
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 - Morphology, DNA barcoding and phylogeny of Macrobiotus persimilis and Macrobiotus polonicus. [Abstract in Atti di Convegno]
Bertolani, Roberto; Cesari, Michele; Lisi, O.; Rebecchi, Lorena; Giovannini, Ilaria; Pilato, G.
abstract

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

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

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

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

2011 - An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens [Articolo su rivista]
Cesari, Michele; Giovannini, Ilaria; Bertolani, Roberto; Rebecchi, Lorena
abstract

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

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

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

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

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

2011 - Filogenesi e integrative taxonomy nei Macrobiotus del “gruppo hufelandi” (Tardigrada, Eutardigrada) [Abstract in Atti di Convegno]
Cesari, Michele; Rebecchi, Lorena; Giovannini, Ilaria; Pilato, G.; Lisi, O.; Vicente, F.; Kiosya, Y.; Bertolani, Roberto
abstract

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

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

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

2011 - 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 - A model study for tardigrade identification [Relazione in Atti di Convegno]
Bertolani, Roberto; Rebecchi, Lorena; Cesari, Michele
abstract

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

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 - Molecular taxonomy and phylogeny of Italian Lepidurus taxa (Branchiopoda: Notostraca) [Articolo su rivista]
Mantovani, B.; Cesari, M.; Scanabissi, F.
abstract

Notostraca taxonomy is controversial, mainly owing to the high intraspecific variability of morphological characters; other complexity factors are represented by the consistent rate of reproductive variation. We undertook the molecular analyses of Italian Lepidurus taxa through the characterization of 12S, 16S and COI mitochondrial genes in new L. apus demes and in the L. couesii populations recently discovered in Southern Italy. For L. apus, the analysis of a wider data set (both in terms of molecular markers and of analysed populations) confirms the previously suggested specific rank of differentiation between its so far recognized subspecies, the Italian L. apus lubbocki and the European L. apus apus. The Italian L. couesii samples pertain to a well-defined taxonomic entity, strictly related to Canadian L. couesii. In 12S dendrograms, this taxon strongly diverges from the other North American Lepidurus species, but the relationships among nearctic taxa are unresolved. On the other hand, a high affinity between Italian/Canadian L. couesii and the circumpolar species L. arcticus is scored and a possible descent from a common ancestor is hypothesized. © 2009 Unione Zoologica Italiana.

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.

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 - Mitochondrial and nuclear DNA variability in the living fossil Triops cancriformis (Bosc, 1801) (Crustacea, Branchiopoda, Notostraca) [Articolo su rivista]
Mantovani, B.; Cesari, M.; Luchetti, A.; Scanabissi, F.
abstract

The living fossil Triops cancriformis comprises bisexual (either gonochoric or hermaphroditic) and unisexual populations. Genetic surveys have recently revealed a general trend of low differentiation of 12S and 16S mitochondrial genes. We, therefore, surveyed further mitochondrial (COI gene and control region) and nuclear markers (dinucleotide microsatellites) to assess the genetic variability and to establish any relationship with the different reproductive modes found in European populations. The mitochondrial analyses confirmed the pattern of low variability. Hence, the low mitochondrial genetic variability appears as a common feature of the genus Triops. The microsatellite analysis found that Italian populations are monomorphic or exhibit little polymorphism, while other European samples display a higher degree of polymorphism and private alleles. Spanish, Austrian and Italian populations show patterns of Hardy-Weinberg disequilibrium that could be explained by the mode of reproduction, or by a higher frequency of null alleles in these populations. The low diversity and differentiation among Italian populations lead us to question the Monopolization Hypothesis. One microsatellite locus appears to be sex-linked, with heterozygotes detected only in males and hermaphrodites. © 2008 Nature Publishing Group All rights reserved.

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.

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 - Genetic variability in European Leptestheria dahalacensis (Rüppel, 1837) (Crustacea, Branchiopoda, Spinicaudata) [Articolo su rivista]
Cesari, M.; Luchetti, A.; Scanabissi, F.; Mantovani, B.
abstract

The genetic variability of the gonochoric Leptestheria dahalacensis (Rüppel, 1837) was studied through the analysis of mitochondrial and nuclear (microsatellite loci) markers in eight Italian and two Central European populations. Mitochondrial data exhibited a low variability, as only six mitotypes were scored: five in Italy and one for both Central European samples, with a very low number of substitutions. All analysed microsatellite loci were variable, with 3-5 alleles per locus and 1-4 alleles per population. All populations were at the Hardy-Weinberg equilibrium, with the exceptions of two samples for locus ldAC-16, due to heterozygote excess, and of four populations for locus ldAC-11, probably linked to the presence of null alleles. A substantial population structuring was found between Central European and Italian samples for both utilized markers. This observation may be explained by isolation by distance and/or recent isolation events. On the other hand, the absence of a clear inter-pond variability in Italian sample comparisons may be ascribed to high dispersal ability in the short range. © 2007 Springer Science+Business Media B.V.

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.

2006 - DNA recovered and sequenced from an almost 7000 y-old Neolithic polypore, Daedaleopsis tricolor [Articolo su rivista]
Bernicchia, A.; Fugazzola, M. A.; Gemelli, V.; Mantovani, B.; Lucchetti, A.; Cesari, M.; Speroni, E.
abstract

Ten fragments of a wood-inhabiting polypore were found in the early Neolithic village of 'La Marmotta' (Anguillara Sabazia, Rome), formerly located on the shore of the Bracciano Lake. Five of these were found in three different huts, and two outside near other structures. Some fragments of the partly degraded specimens were used for DNA sequencing in order to the identity of the fungus, which proved to be Daedaleopsis tricolor. Pharmacological aspects of this and other previously discovered prehistoric polypores are also noted. © 2005 The British Mycological Society.

2006 - Production of intersexes and the evolution of androdioecy in the clam shrimp eulimnadia texana (crustacea, branchiopoda, spinicaudata) [Articolo su rivista]
Weeks, S. C.; Reed, S. K.; Cesari, M.; Scanabissi, F.
abstract

The production of low numbers of offspring that exhibit a mixture of male and female traits (termed “intersexes”) is commonly reported for crustaceans. The production of intersexes has been ascribed to both genetic and non-genetic (e.g., parasitic infections and environmental pollutants) causes. Herein we report on two observed types of intersexes in the clam shrimp Eulimnadia texana: (1) a “morphological” intersex, possessing secondary male characteristics (e.g., claspers) and an eggproducing gonad, and (2) a “gonadal” intersex, possessing primarily male traits (e.g., male secondary sexual characters and male gamete production) but also producing low levels of abortive female gametes. We propose that these intersexes are likely the products of low frequencies of crossing over between the sex determining chromosomes that result in the array of observed mixed sexual phenotypes. Additionally, we suggest that the low-level production of intersexes, combined with the ephemeral nature of the habitats occupied by these shrimp, may explain the preponderance of androdioecy (mixtures of males and hermaphrodites) found in these clam shrimp, and possibly branchiopods more generally. © 2006 Taylor &amp; Francis Group, LLC.

2006 - Ultrastructure of the male gonad and male gametogenesis in the clam shrimp Eulimnadia texana (Crustacea, Branchiopoda, Spinicaudata) [Articolo su rivista]
Scanabissi, F.; Cesari, M.; Reed, S. K.; Weeks, S. C.
abstract

The ultrastructure of the male gonad of Eulimnadia texana (Branchiopoda, Spinicaudata) has been observed for the first time to investigate the sexuality of a well-studied case of androdioecy in the animal kingdom. The male gonad is a double structure located in the hemocoel throughout the entire body length on each side of the midgut. Male gametes originate from the wall and mature centripetally toward the lumen; the proliferative activity is very high and continuous and therefore the mature gonad is full of numerous germ cells. Inside the lumen several degenerative stages are found mixed with sperm cells and spermatids, the latter two being not easily distinguishable because of the slight differences between them. The evolutionary meaning of the degenerative process in E. texana male gametes is difficult to explain, and we propose some hypotheses about its possible role or cause in the studied population: (a) to help build spermatophores, (b) to act as a trophic component for viable sperm, (c) as a manifestation of inbreeding depression, and/or (d) to regulate the number of sperm cells. © 2006, The American Microscopical Society, Inc.

2005 - Androdioecy inferred in the clam shrimp Eulimnadia agassizii (Spinicaudata: Limnadiidae) [Articolo su rivista]
Weeks, S. C.; Posgai, R. T.; Cesari, M.; Scanabissi, F.
abstract

Androdioecy (mixtures of males and hermaphrodites) is a rare mating system in both the plant and animal kingdoms. Androdioecy has been described in three branchiopod species, and is best known from the clam shrimp Eulimnadia texana Packard. Herein we describe sex ratio, genetic and histological evidence from the clam shrimp Eulimnadia agassizii Packard that suggest androdioecy is also found in this species. The E. agassizii population sampled had all-females, and when these females were isolated and allowed to produce eggs, those eggs yielded 100% female offspring in 15 out of 15 cases. Additionally, the originally isolated females proved to be completely homozygous at each of the six allozyme loci scored. The offspring from these isolated females also proved to be homozygous for the same alleles as their parent. Tissue sectioning of the gonad found that the "females" actually produced testicular tissue in the posterior portion of the gonad. Taken together, these data are entirely consistent with those of the androdioecious E. texana, and thus indicate that E. agassizii is also an androdioecious species, bringing the total number of branchiopod species with this form of reproduction to four.

2005 - Chromosomes in sexual populations of notostracan and con-chostracan taxa (crustacea, branchiopoda) [Articolo su rivista]
Ombretta, M.; Cesari, M.; Scanabissi, F.; Mantovani, B.; Eder, E.
abstract

Branchiopods reproductive mechanisms range from gonochorism to unisexuality, passing through an-drodioecy. In order to contribute to still lacking or controversial basic knowledges, we analyzed the karyotype of the main Italian taxa: the Notostracan living fossils Triops cancriformis (parthenogenetic) and Lepidurus apus lubbocki (bisexual), and the Conchostracan Eoleptestheria ticinensis (bisexual). Also one male obtained from a supposedly hermaphroditic Austrian population of T. cancriformis was checked. In T. cancriformis a diploid number of 12 chromosomes is observed in both females and male; this is in line with previous results on other Italian populations, but contrasts with observations on European samples. The richness of normal meiotic pictures indicates that the male specimen is able to produce sperms. L. apus lubbocki shows a diploid number of 10 chromosomes; in male specimens the mispairings during meiosis I could explain the haploid number of 6 chromosomes obtained from diakinetic plates in Palestinian samples of the same subspecies found in literature. Irregular meiotic divisions also highlight the ultrastructurally observed abortive spermatogenesis. Finally, in the presently analyzed population of E. ticinensis, the adult females show 10 chromosomes as diploid number, while males present 10 or 11 elements; in the nauplii, chromosome numbers range from 8 to 12, differing among unrelated individuals, between nauplii produced by the same female and even within the same nauplius. B elements are taken into account. © 2005 Taylor &amp; Francis Group, LLC.

2005 - Male occurrence in Austrian populations of Triops cancriformis (Branchiopoda, Notostraca) and ultrastructural observations of the male gonad [Articolo su rivista]
Scanabissi, F.; Eder, E.; Cesari, M.
abstract

Recent reports of occasional males in Austrian populations of Triops cancriformis have promoted interest in an analysis of the ultrastructure of the male gonad. It appears as a double thick and well-defined tubular structure running along the midgut, inside the hemocoel. It is composed of two portions: the wall and the lumen. The former is made up of germ cells that are widespread and intermixed with somatic cells. The lumen is ∼ 200 μm wide and acts as the collecting site of spermatids and sperm. The germ cells are recognizable by their rounded appearance and by the occurrence of exceptionally long synaptonemal complexes in their nuclei. Their maturation implies a volume elongation and an aggregation in a characteristic "cyst" arrangement. The cysts migrate towards the lumen, where they break open, releasing spermatids and sperm. At the end of this process the lumen is filled with maturing gametes. The sperm have very condensed chromatin nuclei and partially reduced cytoplasm where the most striking features are the axoneme and strewn microtubules together with evident pseudopodia. Our morphological data support the full functionality of males in T. cancriformis. However, further work on gametogenesis, distribution of sexes, and genetic analysis of breeding experiments are needed to reach a fuller understanding of the role of males in middle and northern European populations of this species. © 2005 American Microscopical Society, Inc.

2005 - Taxonomy and phylogeny of European Monochamus species: First molecular and karyological data [Articolo su rivista]
Cesari, M.; Marescalchi, O.; Francardi, V.; Mantovani, B.
abstract

The worldwide distributed genus Monochamus Megerle, 1821 (Coleoptera Cerambicydae) comprises beetles that may become pests of economic importance in conifer stands in the Nearctic and Palearctic Regions. Besides direct damage due to the larval tunnelling habits, they have also been recognized as main vectors of the phytoparasitic nematode Bursaphelenchus xylophilus (Steiner &amp; Buhrer, 1934) (Nematoda Aphelenchoididae). We analysed the complete mitochondrial cytochrome oxidase I gene and a fragment of the small subunit RNA gene sequences (1536 base pairs) in the five European species. These are: Monochamus galloprovincialis (Olivier, 1795), morphologically distinguished in two subspecies M. galloprovincialis galloprovincialis (Olivier, 1795) and M. galloprovincialis pistor (Germar, 1818): Monochamus sutor (Linneus 1758); Monochamus saltuarius (Gebler 1830); Monochamus sartor (Fabricius, 1787) and Monochamus urussovi (Fischer, 1806). For appropriate comparisons, also the Asiatic Monochamus alternatus Hope, 1842 and a Japanese M. saltuarius sample have been analysed. Both genes show an absolute identity between the two subspecies of M. galloprovincialis and a strong affinity between M. sartor and M. urussovi: the morphological subdivisions of the former taxon in two subspecies and of the latter in two entities of specific level are therefore not supported genetically. On the other hand, the Italian and the Japanese samples of M. saltuarius always cluster together in all trees, and for the remaining taxa, no doubt about their rank of specific differentiation emerges from present analyses. From a phyletic point of view, tree topology indicates the Japanese M. alternatus as the most differentiated taxon and the Euroasiatic M. saltuarius as basal to all other strictly European entities. Chromosome analyses show that the diploid autosomal complement ranges from 18 in M. saltuarius to 20 in M. galloprovincialis, and 22 in M. sartor, but a XX-Xyp sex determining system is shared by all analysed taxa. The M. saltuarius karyotype appears as the most primitive from which the others may be derived through Robertsonian fissions. Karyological data therefore agree with molecular analyses in indicating a basal position of Euroasiatic M. saltuarius with respect to the group of European Monochamus taxa; among these, M. galloprovincialis and M. sartor represent two clearly diverging evolutionary units. Furthermore, karyotype analyses substantiate molecular conclusions about the identity between M. galloprovincialis galloprovincialis and M. galloprovincialis pistor.

2004 - Characterization of dinucleotide microsatellite loci in the living fossil tadpole shrimp Triops cancriformis (Crustacea Branchiopoda Notostrace) [Articolo su rivista]
Cesari, M.; Mularoni, L.; Scanabissi, F.; Mantovani, B.
abstract

Five dinucleotide markers were obtained from a microsatellite genomic library of the tadpole shrimp Triops cancriformis. The number of alleles at each locus was low (maximum three alleles) but all loci were polymorphic. The presence of null alleles could not be disregarded. Heterozygosity levels were indicative of self ing in the studied populations whose real sexuality will be further tested using these markers.

2004 - Molecular taxonomy and phylogeny of the 'living fossil' lineages Triops and Lepidurus (Branchiopoda: Notostraca) [Articolo su rivista]
Mantovani, B.; Cesari, M.; Scanabissi, F.
abstract

European Triops cancriformis and Lepidurus apus were analysed for 12S and 16S mitochondrial genes and compared to North American and Japanese taxa. There are no cryptic species among European T. cancriformis populations, which are highly homogeneous in comparison to conspecific Japanese samples. T. cancriformis differs from congeneric taxa all over its range, which can be explained by its antiquity. In contrast, the parapatric subspecies L. apus apus and L. apus lubbocki are morphologically conserved and differ substantially at the mtDNA level. The genetic distance values between them are of the same order of magnitude as those observed between American Lepidurus species. Their subspecific status therefore requires further analysis. L. apus apus is more closely related to a L. arcticus sample from Iceland than to L. apus lubbocki. It is also related to a Canadian L. couesii population. Further analyses of populations from the whole range of L. arcticus and the European range of L. couesii are needed to understand the relationships among these notostracan taxa. When considering the two genera, it is clear that Lepidurus is a well supported monophyletic unit, while Triops is polyphyletic, embodying very divergent taxa.

2003 - Polymerase chain reaction amplification of the Bag320 satellite family reveals the ancestral library and past gene conversion events in Bacillus rossius (Insecta Phasmatodea) [Articolo su rivista]
Cesari, M.; Luchetti, A.; Passamonti, M.; Scali, V.; Mantovani, B.
abstract

Polymerase chain reaction amplifications of genomic DNA in 17 individuals of bisexual and parthenogenetic populations of three subspecies of Bacillus rossius (Insecta Phasmatodea) revealed that the species still harbours the whole variability of the ancestral Bag320 satellite family, since monomers of all non-hybrid Bacillus taxa plus private sequences occur in it. Bag320 monomers had not been rescued as a major satellite component in B. rossius, but possibly represent the remnant of a set of diverging sequences present in the Bacillus ancestor. Following the library hypothesis, these monomer variants have been differently amplified along the evolutionary pathways leading to present taxa in agreement with the mitochondrial phylogeny of the genus. The putative converted tracts observed are explained as the results of past gene conversion events. © 2003 Elsevier Science B.V. All rights reserved.

2003 - Unisexuality and molecular drive: Bag320 sequence diversity in Bacillus taxa (Insecta Phasmatodea) [Articolo su rivista]
Luchetti, A.; Cesari, M.; Carrara, G.; Cavicchi, S.; Passamonti, M.; Scali, V.; Mantovani, B.
abstract

Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process. Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B. grandii) to automictic (B. atticus) and apomictic (B. whitei = rossius/grandii; B. lynceorurn = rossius/grandii/atticus) unisexuality. Thelytokous reproduction clearly allows uncoupling of homogenization from fixation. Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B. rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies. For unisexuals, allozymic and mitochondrial clones were also taken into account. Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B. grandii roaretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B. whitei and B. lynceorum. Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B. lynceorum. On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features. Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits.

2002 - Lysiphlebus testaceipes on Aphis gossypii: Studies on remote host discrimination [Articolo su rivista]
Medrzycki, P.; Cesari, M.; Maini, S.
abstract

The ability of Lysiphlebus testaceipes (Cresson) females to discriminate between parasitised and unparasitised Aphis gossypii Glover, and the mechanisms of host discrimination was studied. By comparing the resting time of parasitoid females on leaves with parasitised and unparasitised aphids their ability to host discriminate was evidenced. Y-tube olfactometer tests and direct observations of the searching behaviour of L. testaceipes females showed that the markers released during the parasitisation (at the insertion of the ovipositor and during egg laying) are perceived neither over long nor over short distance, but they are probably responsible of contact host discrimination. However, the parasitoids use their antennae to perceive olfactory cues and thus recognise parasitised hosts. The cues that act over short distance are directly or indirectly induced by the egg or the larva of the parasitoid developing inside the host.