Two new species of Tardigrada from moss cushions (Grimmia sp.) in a xerothermic habitat in northeast Tennessee (USA, North America), with the first identification of males in the genus Viridiscus.

BACKGROUND: The phylum Tardigrada consists of over 1,300 species that inhabit terrestrial, freshwater and marine environments throughout the world. In terrestrial habitats they live primarily in mosses, lichens, leaf litter and soil, whereas tardigrades in freshwater and marine environments are mainly found in sediments and on aquatic plants. More than 65 species have been previously reported in the state of Tennessee, USA. METHODS: Tardigrades present in moss cushions (Grimmia sp.) collected from a xerothermic habitat on the East Tennessee State University campus, Johnson City, TN, USA, were extracted, mounted on slides, identified, and counted. Additional samples of fresh dried moss were used for integrative analyses, including morphological analysis with phase contrast (PCM) and scanning electron microscopy (SEM), as well as molecular analyses of COI, 18S rRNA, 28S rRNA, and ITS-2 of the Macrobiotus and Milnesium species. RESULTS: Five species were found, including two species new to science: Viridiscus miraviridis sp. nov. and Macrobiotus basiatus sp. nov. Viridiscus miraviridis sp. nov. differs from other members of the genus mainly by having a different type of dorsal cuticle and some other, more subtle, morphometric characters. In addition to the two new species, Viridiscus perviridis and Viridiscus viridissimus were present, and males of Vir. viridissimus were found for the first time, the first record of males in the genus Viridiscus. Macrobiotus basiatus sp. nov. is most similar to Macrobiotus nelsonae, but it differs from Mac. nelsonae mainly by the stylet supports being situated in a more anterior position, shorter and narrower egg processes, and a smaller number of areoles around the egg processes. Moreover, the identification of Milnesium inceptum was confirmed as the first record for the USA by analysis of COI.

Integrative description of five Pseudechiniscus species (Heterotardigrada: Echiniscidae: the suillus-facettalis complex).

Pseudechiniscus is a morphologically homogeneous heterotardigrade genus with a relatively low number of morphological features useful for the species discrimination. The species of the Pseudechiniscus suillus-facettalis complex are some of the most challenging tardigrades to identify. Here, we examine several populations from Antarctica, Italy, Madagascar and Norway that would have most likely been attributed to Pse. suillus prior to the recent redescription of the species. Populations were analysed using integrative taxonomy-a combination of classical morphology and morphometry, as well as genetic data. Besides minute differences in dorsal sculpture and morphometry, we found clear, species-specific differences in ventral sculpture which are very useful in discrimination of Pseudechiniscus species. Based on morphology (mainly ventral sculpture) and significant genetic distances in COI and ITS-2 sequences, we describe five new species: Pse. angelusalas sp. nov. from Madagascar, Pse. dastychi sp. nov. from Antarctica, Pse. ehrenbergi sp. nov. from Italy as well as Pse. indistinctus sp. nov. and Pse. lacyformis sp. nov. from Norway. Finally, we provide an updated phylogenetic tree of the genus Pseudechiniscus based on COI sequences.

Two new species of the genus Milnesium Doyère, 1840 (Tardigrada, Apochela, Milnesiidae) from Madagascar.

The knowledge of the diversity and distribution of tardigrades on Madagascar is rather poor. To date, only 13 tardigrade taxa have been reported from this region (including one Milnesium species). We examined 46 specimens belonging to two new-to-science species of the genus Milnesium described herein using an integrative approach, including classical morphology and molecular marker (COI, ITS-2 and 28S rRNA) analysis. The species were found in two moss and lichen samples collected in the Ivohibory forest in Fianarantsoa Province. Milnesium matheusi sp. nov., with claw configuration [3-3]-[3-3] and rather wide buccal tube, morphologically is most similar to: Mil. beatae Roszkowska, Ostrowska & Kaczmarek, 2015, Mil. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014, Mil. eurystomum Maucci, 1991, Mil. shilohae Meyer, 2015 and Mil. tumanovi Pilato, Sabella & Lisi, 2016; however, it differs from these by morphometric characteristics. Milnesium wrightae sp. nov., by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919. However, Mil. wrightae sp. nov. differs from Mil. quadrifidum by claw configuration ([4-4]-[4-4] in Mil. quadrifidum vs. [3-3]-[4-4] in Mil. wrightae sp. nov.), but also by the position of the fourth points on secondary branches of claws IV, which are located near the base of the claw in the new species and near the top of the claw in Mil. quadrifidum. Genotypic analysis showed that Mil. matheusi sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. t. tardigradum (ITS-2) while Mil. wrightae sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. matheusi (ITS-2). Five Milnesium taxa are recorded from the African region, including the two new species from Madagascar reported in this study.

Variability of Echiniscus tristis Gasiorek & Kristensen, 2018-is morphology sufficient for taxonomic differentiation of Echiniscidae?

The majority of species in the genus Echiniscus (Heterotardigrada) have been described based on differences in the chaetotaxy or dorsal sculpture. Dorsal sculpture is, in general, considered to be species-specific and not very variable; however, many problems have arisen due to various interpretations of microscope images, which has led to taxonomic confusion in the genus Echiniscus. Conversely, chaetotaxy is generally much easier to interpret, even using low-quality microscope optics. In this study, we emended the description of Madagascan population of Echiniscus tristis Gasiorek & Kristensen, 2018 that exhibits several different types of chaetotaxy and dorsal sculpture. The analysed specimens were characterised by two types of chaetotaxy, A-C-Dd-E and A-Dd-E, but we also found a wide range of variation in appendage number, shape and length. The observed differences are partly correlated with life stages. Additionally, we analysed DNA sequences of 28S rRNA, ITS-2 and COI of the two main morphotypes, and did not find significant genetic differentiation of the two morphotypes. This highlights the importance of analysing the morphology of both immature stages and adults, as well as of DNA markers in tardigrade species identification.

Human VDAC isoforms differ in their capability to interact with minocycline and to contribute to its cytoprotective activity.

It has been previously demonstrated that cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H2O2 requires the presence of functional VDAC (YVDAC1). Thus, we decided to transform YVDAC1-depleted yeast cells (Δpor1 cells) with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their involvement in the minocycline cytoprotective effect. We observed that only expression of HVDAC3 in Δpor1 cells provided minocycline-mediated cytoprotection against H2O2 although all human isoforms are functional in Δpor1 cells. The observation appears to be important for on-going discussion concerning VDAC isoform roles in mitochondria and cell functioning.

Putative new groups of invertebrate water channels based on the snail Helix pomatia L. (Helicidae) MIP protein identification and phylogenetic analysis.

Water channel proteins, classified as a family of Membrane Intrinsic Proteins (MIPs) superfamily, enable rapid movement of water and small uncharged molecules through biological membranes. Although water channel proteins are required in several important processes characteristic for the animals, such as osmoregulation, mucus secretion, or defense against desiccation, molluscs, until now, have been very poorly explored in this aspect. Therefore, we decided to study MIPs in Helix pomatia L. applied as a model in studies on terrestrial snail physiology. Our studies consisted in: the snail organ transcriptome sequencing and consecutive bioinformatic analysis of the predicted protein, estimation of the encoding transcript expression (qPCR), investigation of the predicted protein function in the yeast Saccharomyces cerevisiae cells, and the phylogenetic analysis. We identified six water channel proteins, named HpAQP1 to HpAQP6. All of them were proven to transport water, two of them (HpAQP3 and HpAQP4) were also shown to be able to transport glycerol, and other two (HpAQP5 and HpAQP6) to transport H2O2. Phylogenetic analysis indicated that the proteins either fell into aquaporins (HpAQP1, HpAQP2 and HpAQP5) or formed new groups of invertebrate water channel proteins, not described until now, that we suggest to term malacoglyceroporins (HpAQP3 and HpAQP4) and malacoaquaporins (HpAQP6). Thus, the classification of animal water channels based on the vertebrate proteins and including aquaporin, aquaglyceroporin, S-aquaporin and AQP8-type grades does not reflect diversity of these proteins in invertebrates. The obtained results provide important data concerning diversity of water channel protein repertoire in aquatic and terrestrial invertebrates and should also contribute to the improvement of animal water channel classification system.

The Association of VDAC with Cell Viability of PC12 Model of Huntington's Disease.

It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington's disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application.