How long can tardigrades survive in the anhydrobiotic state? A search for tardigrade anhydrobiosis patterns.

Anhydrobiosis is a desiccation tolerance that denotes the ability to survive almost complete dehydration without sustaining damage. The knowledge on the survival capacity of various tardigrade species in anhydrobiosis is still very limited. Our research compares anhydrobiotic capacities of four tardigrade species from different genera, i.e. Echiniscus testudo, Paramacrobiotus experimentalis, Pseudohexapodibius degenerans and Macrobiotus pseudohufelandi, whose feeding behavior and occupied habitats are different. Additionally, in the case of Ech. testudo, we analyzed two populations: one urban and one from a natural habitat. The observed tardigrade species displayed clear differences in their anhydrobiotic capacity, which appear to be determined by the habitat rather than nutritional behavior of species sharing the same habitat type. The results also indicate that the longer the state of anhydrobiosis lasts, the more time the animals need to return to activity.

Experimental evidence for snails dispersing tardigrades based on Milnesium inceptum and Cepaea nemoralis species.

Dispersal abilities in animals contribute to their local genetic variability and species persistence. However, the mechanisms facilitating a short-distance migration of small organisms remain underexplored. In this study we experimentally tested the role of land snails for a fine-scale transmission of tardigrades. We also check the ecological relationship between these two groups, by testing the impact of snail's mucus on tardigrades in anhydrobiosis. All the experiments were conducted under laboratory conditions. As model organisms, we used a tardigrade species Milnesium inceptum and a snail species Cepaea nemoralis. The selection of the experimental animals was dictated by their co-occurrence in natural habitats and similar atmospheric conditions required for them to remain active. Results of our experiments support the assumption that snails may transfer active tardigrades for short distances. On the other hand, the effect of the snails mucus on tardigrade recovery to active life after anhydrobiosis was negative. Death rates of tardigrades in anhydrobiosis (tun) were higher when affected by mucus compared to mucus-free tuns.

Verification of Hypsibius exemplaris Gasiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research.

Anhydrobiosis is considered to be an adaptation of important applicative implications because it enables resistance to the lack of water. The phenomenon is still not well understood at molecular level. Thus, a good model invertebrate species for the research is required. The best known anhydrobiotic invertebrates are tardigrades (Tardigrada), considered to be toughest animals in the world. Hypsibius. exemplaris is one of the best studied tardigrade species, with its name "exemplaris" referring to the widespread use of the species as a laboratory model for various types of research. However, available data suggest that anhydrobiotic capability of the species may be overestimated. Therefore, we determined anhydrobiosis survival by Hys. exemplaris specimens using three different anhydrobiosis protocols. We also checked ultrastructure of storage cells within formed dormant structures (tuns) that has not been studied yet for Hys. exemplaris. These cells are known to support energetic requirements of anhydrobiosis. The obtained results indicate that Hys. exemplaris appears not to be a good model species for anhydrobiosis research.

The tardigrade Hypsibius exemplaris has the active mitochondrial alternative oxidase that could be studied at animal organismal level.

Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens' tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals' behavior observation as well as by measurement of intact specimens' whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.

Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: is there molecular evidence for its presence in tardigrades?

We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e., a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in previous studies on the microbial community of tardigrades, and Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.

An integrative description of Diploechiniscus oihonnae/ (Richters, 1903) population from near the original type locality in Merok (Norway).

In the present study, we used integrative taxonomy to describe a population of Diploechiniscus oihonnae (Richters, 1903) from the neotype locality in Merok (Norway). We found no differences in the chaetotaxy formula between the life stages and sex of Dpl. oihonnae. The presence of filaments at Bd in some specimens of Dpl. oihonnae appears to be random and most likely represents the morphological variability of this species. We also obtained DNA sequences of 18S rRNA, 28S rRNA, ITS-2, and COI of Dpl. oihonnae from the neotype locality for comparison with the sequences available in GenBank, which showed low genetic differences between the neotypic population and specimens from other localities. Therefore, we decided to establish our specimens from Merok as neotype and neoparatypes of Dpl. oihonnae. Additionally, based on morphological characters, Dpl. horningi (Schuster Grigarick, 1971) was synonymised with Dpl. oihonnae.

Mitochondrial alternative oxidase contributes to successful tardigrade anhydrobiosis.

Anhydrobiosis can be described as an adaptation to lack of water that enables some organisms, including tardigrades, to survive extreme conditions, even some that do not exist on Earth. The cellular mechanisms underlying anhydrobiosis are still not completely explained including the putative contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX), described as a drought response element in plants, was recently proposed for various invertebrates including tardigrades, we investigated whether AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its contribution to the tardigrade revival after anhydrobiosis of different durations. We observed that AOX activity was particularly important for M. inceptum revival after the long-term tun stage but did not affect the rehydration stage specifically. The results may contribute to our understanding and then application of anhydrobiosis underlying mechanisms.

Tolerance to Anhydrobiotic Conditions Among Two Coexisting Tardigrade Species Differing in Life Strategies.

Water availability is one of the most important factors for terrestrial life. Terrestrial habitats may periodically become dry, which can be overcome by an organism's capability to undergo anhydrobiosis. In animals, this phenomenon has been reported for invertebrates, with tardigrades being the best-known. However, different tardigrade species appear to significantly differ in their anhydrobiotic abilities. While several studies have addressed this issue, established experimental protocols for tardigrade dehydration differ both within and among species, leading to ambiguous results. Therefore, we apply unified conditions to estimate intra-and interspecies differences in anhydrobiosis ability reflected by the return to active life. We analysed Milnesium inceptum and Ramazzottius subanomalus representing predatory and herbivorous species, respectively, and often co-occur in the same habitat. The results indicated that the carnivorous Mil. inceptum displays better anhydrobiosis survivability than the herbivorous Ram. subanomalus. This tendency to some degree coincides with the time of "waking up" since Mil. inceptum showed first movements and full activity of any first individual later than Ram. subanomalus. The movements of all individuals were however observed to be faster for Mil. inceptum. Differences between the experimental groups varying in anhydrobiosis length were also observed: the longer tun state duration, the more time was necessary to return to activity.

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 genera of long clawed Isohypsibioidea Guil, Jørgensen amp; Kristensen, 2019.

Two new eutardigrade genera Vladimirobius gen. nov. and Weglarskobius gen. nov. are erected for the species Grevenius irregibilis (Biserov, 1992) and Isohypsibius altai Kaczmarek Michalczyk, 2006a, respectively. They are characterized by strongly modified claws, bucco-pharyngeal apparatus of the Isohypsibius variant, pharyngeal apophyses, and placoids present. The primary branches of internal claws of Vladimirobius gen. nov. are wide in lateral view and have very prominent gibbosity-like projections, whereas primary branches of external claws are thin and extremely long. In Weglarskobius gen. nov. claws IV are similar in shape and size, but the primary branches are extremely long, and thin. Moreover, claws on legs I-III vary greatly in size and shape in Weglarskobius gen. nov. Such supposed autapomorphies fully justify erection of the two new genera, even if the genetic data are presently unavailable.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Current knowledge on Mexican tardigrades with a description of Milnesium cassandrae sp. nov. (Eutardigrada: Milnesiidae) and discussion on the taxonomic value of dorsal pseudoplates in the genus Milnesium Doyère, 1840.

Mexico is a megadiverse country, however its tardigrade fauna is poorly known and to date 44 species have been reported from this region. In the present paper, an update of the reported Mexican tardigrades is presented, increasing the number of recorded tardigrades to 55 species (11 new records). Moreover, we describe Milnesium cassandrae sp. nov. from State of Nuevo León in northern Mexico. The new species is most similar to Mil. krzysztofi Kaczmarek Michalczyk, 2007 and Mil. beasleyi Kaczmarek, Jakubowska Michalczyk, 2012, but it differs from them mainly by details of the dorsal sculpture and some morphometric characters. We also discuss the taxonomic value of a recently described feature, 'pseudoplates', for differentiating the taxa within the genus Milnesium Doyère, 1840. [Zoobank URL: urn:lsid:zoobank.org:act:0684AE18-0510-4F7B-B75D-AE5177FBF2A2].

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.

Two new species of Tardigrada from Ecuador (South America).

In two moss samples collected in El Cajas National Park (Azuay Province, Ecuador), 33 specimens and 19 eggs of a new species of Macrobiotus and 56 heterotardigrades belonging to a new Echiniscus species were extracted. Macrobiotus dulciporus sp. nov. belongs to the hufelandi group and is most similar to Macrobiotus maculatus Iharos, 1973. It differs from M. maculatus mainly based on the presence of an oral cavity armature of the patagonicus type, smooth egg chorion surface, lower number of processes on the egg circumference and some morphometric characters of the egg processes. Echiniscus hannae sp. nov. belongs to the bigranulatus group, but differs from all species in this group due to the presence of large, smooth areas on its dorsal plate.

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.

Tardigrade indexing approach on exoplanets.

Finding life on other worlds is a fascinating area of astrobiology and planetary sciences. Presently, over 3800 exoplanets, representing a very wide range of physical and chemical environments, are known. Scientists are not only looking for traces of life outside Earth, but they are also trying to find out which of Earth's known organisms (ex: tardigrades (water bears)) would be able to survive on other planets. In our study, we have established a metric tool for distinguishing the potential survivability of active and cryptobiotic tardigrades on rocky-water and water-gas planets in our solar system and exoplanets, taking into consideration the geometrical means of six physical parameters such as radius, density, escape velocity, revolution period, surface temperature, and surface pressure of the considered planets. More than 3800 exoplanets are available as the main sample from Planetary Habitable Laboratory - Exoplanet Catalog (PHL-EC), from which we have chosen 57 exoplanets in our study including Earth and Mars, with water composition as reference. The Active Tardigrade Index (ATI) and Cryptobiotic Tardigrade Index (CTI) are two metric indices with minimum value 0 (= tardigrades cannot survive) and maximum 1 (= tardigrades will survive in their respective state). Values between 0 and 1 indicate a percentage chance of the active or cryptobiotic tardigrades surviving on a given exoplanet. Among known planets some of the exoplanets are tabulated as ATI and CTI indices for sample representation like: Kepler-100d, Kepler-48d, Kepler-289b, TRAPPIST-1 f and Kepler-106e. The results with Mars as the threshold indicates that Mars could be the only rock-water composition planet that could be more suitable for tardigrades than other considered exoplanets.

An integrative description of a new tardigrade species Mesobiotus romani sp. nov. (Macrobiotidae: harmsworthi group) from the Ecuadorian Pacific coast.

In a mixed moss and lichen sample collected in Esmeraldas Province in north-western Ecuador, 20 tardigrades and 11 eggs, belonging to a new species of the genus Mesobiotus, were found. In addition to the traditional taxonomic description with morphometrics, light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragments of the new species. Based on the egg chorion morphology, Mesobiotus romani sp. nov. is the most similar to: M. binieki, M. coronatus, M. dimentmani, M. patiens, M. perfidus, M. philippinicusi, M. pseudoblocki, M. pseudocoronatus, M. pseudopatiens, M. radiatus, M. rigidus, M. simulans and M. wuzhishanensis, but differs mainly by some specific characters of both egg and adult morphology, and morphometrics.

An integrative redescription of the nominal taxon for the Mesobiotus harmsworthi group (Tardigrada: Macrobiotidae) leads to descriptions of two new Mesobiotus species from Arctic.

The Mesobiotus harmsworthi group has a global distribution, with localities in polar, temperate and tropical zones. Since the first species of the harmsworthi group was described in the beginning of the 20th century, tens of new species within the group were found and named. However, the diagnosis of the nominal Mesobiotus harmsworthi is insufficient and enigmatic, thus it can be is a serious obstacle in solving the taxonomy of this group. Here, we integratively redescribe the nominal species for the genus Mesobiotus, i.e., Mesobiotus harmsworthi and clarify taxonomic statuses of the two subspecies: M. harmsworthi harmsworthi and M. harmsworthi obscurus that have been recognised as distinct taxa for more than three decades. Traditionally, egg chorion in M. harmsworthi was considered almost smooth and without any traces of areolation, however here we report many misunderstandings that accumulated across decades and we show that, in fact, the chorion in this species exhibits a partially developed areolation. We present an integrative (morphological, morphometric and molecular) diagnosis of the nominal taxon and we confirm that it differs from other species of the harmsworthi group by morphological characters of both animals and eggs. Additionally, we describe two new species of the genus Mesobiotus: M. skorackii sp. nov. from the Kyrgyz Republic (using classical morphological description) and M. occultatus sp. nov. from Svalbard Archipelago (by means of integrative taxonomy). Finally, we also provide the first genetic phylogeny of the genus Mesobiotus based on COI sequences which, together with molecular species delimitation, independently confirms the validity of the analysed taxa.

Tardigrades from Nahuel Huapi National Park (Argentina, South America) with descriptions of two new Macrobiotidae species.

In 31 samples of mosses and lichens collected in the Argentinean province of Río Negro, 657 tardigrades, 53 exuviae and 219 free-laid eggs were found. In total, 20 species were identified: Diphascon chilenense, Dip. mitrense, Echiniscus bigranulatus, Ech. corrugicaudatus, Ech. merokensis merokensis, Ech. testudo, Hebesuncus mollispinus, Hypsibius convergens, Macrobiotus cf. anderssoni, Mac. andinus, Mac. kazmierskii, Mac. patagonicus, Mesobiotus szeptyckii, Mes. pseudoblocki sp. nov., Milnesium argentinum, Mil. beatae, Mil. brachyungue, Mil. granulatum, Mopsechiniscus granulosus, Minibiotus pseudostellarus sp. nov. Of the two new species, Mesobiotus pseudoblocki sp. nov. is most similar to Mes. blocki, but it differs mainly by the lack of dentate lunules, smaller eggs and presence of reticular design on egg processes. Minibiotus pseudostellarus sp. nov. is most similar to Min. constellatus, Min. eichhorni, Min. sidereus or Min. vinciguerrae, but it differs from them by the presence of 'pseudo-star'-shaped pores in the dorsal cuticle instead of fully developed 'stars' and by other morphometric characters.