Welcome by Professor Roberto Bertolani

Dear All, I wish also extend the greetings and best wishes for a successful meeting from Lorena Rebecchi and Roberto Guidetti, University of Modena and Reggio Emilia, co-organizers of the Symposium. I am very glad to meet you in Catania, some of you again, some others for the first time. Thank you to all participants. A particular welcome to Diane, the only person who shares with me participation to all ten Symposia. I hope that this meeting may be an important occasion for an exchange of information, ideas, suggestions and debates among researchers who use tardigrades as a model, but are also interested in general and stimulating problems of animal biology

A model study for tardigrade identification

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 morphological 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

Tardigradi della Sardegna e di alcune piccole isole circum-sarde.

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

High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae)

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\u201321.8%). 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

Energy allocation in two species of Eutardigrada

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

Stress response of a boreo-alpine species of tardigrade, Borealibius zetlandicus (Eutardigrada, Hypsibiidae)

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

A DNA barcoding approach in the study of tardigrades

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”

An integrated study of the biodiversity within the Pseudechiniscus suillus-facettalis group (Heterotardigrada:Echiniscidae):Echiniscidae)

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 \u2018suillus\u2013facettalis group\u2019, 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\u2013facettalis group lineage. Inside the P. suillus\u2013facettalis 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