Roberto Guidetti

Long‐term anhydrobiotic survival in semi‐terrestrial micrometazoans

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

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.

Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.

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.

A pest alien invasion in progress: potential pathways of origin of the brown marmorated stink bug Halyomorpha halys populations in Italy

Abstract The 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.

Survival and DNA degradation in anhydrobiotic tardigrades.

SUMMARY 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 the anhydrobiotic tardigrade Paramacrobiotus richtersi as a model organism, 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 blotting 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, and (ii) high temperatures and high RH values change the state of the disaccharide trehalose, reducing its protective role.

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.

The morphological and molecular analyses of a new South American urban tardigrade offer new insights on the biological meaning of the Macrobiotus hufelandi group of species (Tardigrada: Macrobiotidae)

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. http://www.zoobank.org/urn:lsid:zoobank.org:pub:2CA51ABA-5570-4AFB-8182-CE499D97B0F1