Roberto Bertolani

A revision of the Macrobiotus hufelandi group (Tardigrada, Macrobiotidae), with some observations on the taxonomic characters of eutardigrades

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

Tardigrade Resistance to Space Effects: First Results of Experiments on the LIFE-TARSE Mission on FOTON-M3 (September 2007)

The Tardigrade Resistance to Space Effects (TARSE) 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. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.

Phylogeny of Eutardigrada: New molecular data and their morphological support lead to the identification of new evolutionary lineages

An extensive study of the phylogeny of Eutardigrada, the largest class of Tardigrada, has been performed analyzing one hundred and forty sequences (eighty of which newly obtained) representative of one hundred and twenty-nine specimens belonging to all families (except Necopinatidae) of this class. The molecular (18S and 28S rRNA) results were compared with new and previous morphological data, allowing us to find new phylogenetic relationships, to identify new phylogenetic lineages, to erect new taxa for some lineages, and to find several morphological synapomorphies supporting the identified clusters. The class Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies Macrobiotoidea, Hypsibioidea, Isohypsibioidea, and Eohypsibioidea, and all the families and subfamilies considered, although with emended diagnoses in several cases. In addition, new taxa have been erected: the new subfamily Pilatobiinae (Hypsibiidae) with the new genus Pilatobius, as well as an upgrading of Diphascon and Adropion to genus level, previously considered subgenera of Diphascon. Our results demonstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative and comparative approach using both molecular and morphological data is necessary to better elucidate evolutionary relationships.

Facts and fiction about long-term survival in tardigrades

Tardigrades have achieved a widespread reputation for an ability to survive more than a century in an inactive, ametabolic state called cryptobiosis. However, a closer look at the empirical evidence provides little support for the claim that tardigrades are capable of century-long survival. Instead, current evidence suggests that a decade may more realistically represent the upper limit of cryptobiotic survival in the most resistant tardigrades.

Phylogenetic analysis of Macrobiotidae (Eutardigrada, Parachela): a combined morphological and molecular approach

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

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae).

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 & 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 closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I 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.

Form and function of the feeding apparatus in Eutardigrada (Tardigrada)

Tardigrade feeding apparatus is a complex structure with considerable taxonomic significance that can be schematically divided into four parts: buccal ring, buccal tube, stylet system, and pharynx. We analyzed the fine morphology and the tridimensional organization of the tardigrade buccal–pharyngeal apparatus in order to clarify the relationships between form and function and to identify new characters for systematic and phylogenetic studies. We conducted a comparative analysis of the cuticular structures of the buccal–pharyngeal apparatuses of twelve eutardigrade species, integrating data obtained by SEM and LM observations. Morphological diversity was observed and new cuticular structures such as the stylet coat of the stylet system were identified. The synthesis of the buccal–pharyngeal apparatus during molting was also analyzed obtaining a clear developmental sequence of its resynthesis. These findings lead us to redefine the previous interpretations of the functioning mechanisms of the buccal–pharyngeal apparatus and provide a more specific relationship between tardigrade diet and the anatomy of their feeding apparatuses. In addition, the detection by energy-dispersive X-ray spectroscopy of calcium in the stylets, buccal tube, and placoids of eutardigrade species (i.e., Milnesium tardigradum, Paramacrobiotus richtersi) indicates that CaCO3 incrustations are not an exclusive feature of heterotardigrades and lead to suppose that this trait was present in the ancestors of both classes.

Evolution of the Reproductive Mechanisms in Tardigrades — A Review

Abstract Although tardigrades can reproduce only through gametes they have exploited several modes of reproduction, which may be determined by their environment. Marine species (mainly heterotardigrades) are gonochoristic; hermaphroditism is only cited once, and parthenogenesis is unknown. In many cases females mature one egg at a time throughout adult life, whereas males are semelparous. Gonochorism is still present in limno-terrestrial species, while sporadic hermaphroditism occurs in several eutardigrade families. Thelytoky is the most common mode of reproduction in non-marine Tardigrada. Females are iteroparous, laying groups of eggs (free or in the exuvium), while males are semelparous (in a limnic species) or iteroparous with a continuous or cyclical maturation of the spermatozoa (in species from moss and leaf litter). Self-fertilisation appears to characterise hermaphroditic species, found in freshwater, mosses, leaf litter and soil. Egg maturation in these species is similar to that of the gonochoristic species, while spermatozoa mature in appreciable numbers before the oocytes, subsequently maturing continuously but in small numbers over the life of the animal. Parthenogenesis in limno-terrestrial tardigrades always appears continuous. In many species only females occur, but morpho-species populations may be found with both bisexual amphimictic (diploid) and unisexual thelytokous (often but not always polyploid) cytotypes. We can hypothesise that with the evolution of cryptobiosis and passive dispersal unstable and isolated habitats may favour parthenogenesis and self-fertilisation, as both reproductive modes allow colonisation of a new territory by a single individual. Parthenogenesis and hermaphroditism do not occur in the same species, and we can surmise that self-fertilisation will only evolve where parthenogenesis has never occurred.

Maturative pattern of ovary and testis in eutardigrades of freshwater and terrestrial habitats

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

Diapause in tardigrades: a study of factors involved in encystment

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