Università degli studi di Modena e Reggio Emilia

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

Long-term environment acidifications due to decrease pH of the rainwaters affect both soils and water bodies. The organisms most likely to be affected by acid rain are the ones that possess vital organs made of calcium carbonate; among them are tardigrades, presenting aragonite piercing stylets in feeding apparatuses. A positive relationship between acidic rainfall and loss of tardigrades diversity has been already shown, but there is lack of knowledge of its lethal and sublethal effects. This study quantifies the effects of the acute exposure of three eutardigrade, Acutuncus antarcticus, Hypsibius exemplaris, and Macrobiotus cf. hufelandi, to synthetic acid rains and to organic and inorganic acids (hydrochloric, acetic, sulfuric, and nitric acids) naturally occurring in the environment. The cumulative proportion of dead animals in respect of exposition time was fitted to cumulative Weibull Distribution using a Bayesian framework. At the end of the experiments, animals were observed to investigate damages to their piercing stylets. Besides, stylets were finely morphologically described with Scanning Electron Microscopy. This study shows that acid rains and the other tested acids negatively affect tardigrades accordingly with pH, time of exposure, and tardigrade species. Freshwater species show a better resistance to acidity than the moss dwelling species, which can better acclimate over the time to low pH. The stylets resulted unaltered in almost all of the alive specimens. The results suggest that the tested tardigrades taxa have the ability to buffer the environmental proton change and the negative effect on their populations could be counteracted.

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.

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.

Species checklists are powerful and important tools of communication between taxonomists and applied environmental biologists, which in turn lead to well-planned and successful conservation strategies and ecological studies. Despite this, only recently the interest on compiling systematic checklists is growing among taxonomists who study tardigrades - micrometazoans that inhabit almost every habitat worldwide. As the Finnish records of tardigrades (a.k.a. water bears) species are incomplete, outdated and no checklist has ever been compiled for this country, an easy-to-consult checklist is here reported. This checklist covers all Finnish tardigrade taxa identified in the past and in the 13 samples collected for this study. A total of 68 tardigrade species are recorded from Finland, with 6 of them being new records presented in this contribution. Of these species, four have their loci tipici in Finland and we provide an English translation of their original Gennan descriptions. A Generalised Linear Model was used to test the effect of sampling effort and area size on the number of species recorded in each biogeographical province of Finland. The results showed that geographical differences in species richness can be explained solely by sampling effort. The number of tardigrade species recorded in Finland corresponds to about 5% of all described species in the phylum, thus indicating a potential high richness for this country. However, the results of the Generalised Linear Model highlight that a reliable knowledge of the tardigrade diversity in Finland will be reached only with a more uniform and intensive sampling effort.

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.