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Honey bees (Apis mellifera) are one of the most crucial pollinators, providing vital ecosystem services. Their development and functioning depend on essential nutrients and substances found in the environment. While collecting nectar as a vital carbohydrate source, bees routinely encounter low doses of ethanol from yeast fermentation. Yet, the effects of repeated ethanol exposure on bees' survival and physiology remain poorly understood. Here, we investigate the impacts of constant and occasional consumption of food spiked with 1% ethanol on honey bee mortality and alcohol dehydrogenase (ADH) activity. This ethanol concentration might be tentatively judged close to that in natural conditions. We conducted an experiment in which bees were exposed to three types of long-term diets: constant sugar solution (control group that simulated conditions of no access to ethanol), sugar solution spiked with ethanol every third day (that simulated occasional, infrequent exposure to ethanol) and daily ethanol consumption (simulating constant, routine exposure to ethanol). The results revealed that both constant and occasional ethanol consumption increased the mortality of bees, but only after several days. These mortality rates rose with the frequency of ethanol intake. The ADH activity remained similar in bees from all groups. Our findings indicate that exposure of bees to ethanol carries harmful effects that accumulate over time. Further research is needed to pinpoint the exact ethanol doses ingested with food and exposure frequency in bees in natural conditions., (© 2024. The Author(s).)

Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance., (© 2024 John Wiley & Sons Ltd.)

The tardigrade genus Acutuncus has been long thought to be an Antarctic endemism, well adapted to this harsh environment. The Antarctic endemicity of Acutuncus was recently dispelled with the description of Acutuncus mariae Zawierucha, 2020 found in the Svalbard archipelago. The integrated analyses on two newly found Acutuncus populations from UK and Italy, and a population of Acutuncus antarcticus found close to its type locality allowed us to expand the climatic and geographic range of the genus Acutuncus. These findings also allowed us to re-evaluate the morphological diagnoses of Acutuncus and accommodate it in the newly proposed monotypic family Acutuncidae fam. nov. Two new Acutuncus species morpho-groups are instituted based on eggs morphology: one (Acutuncus antarcticus morphogroup) including the Antarctic Acutuncus taxa characterized by eggs with long pillars within the chorion and eggs laid freely to the environment, the other (Acutuncus mariae morphogroup) including the European species, characterized by eggs with short pillars within the chorion and eggs laid in the exuvium. Finally, we describe two new Acutuncus species from Europe: Acutuncus mecnuffisp. nov. and Acutuncus giovanniniaesp. nov., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In a moss sample collected in Ribeiro Frio, Madeira, Paramacrobiotus gadabouti sp. nov. was found and described using the integrative taxonomy approach. The new species is described based on morphological and morphometric data from both phase-contrast light microscopy (PCM), as well as scanning electron microscopy (SEM). Moreover, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) markers, were used to elucidate the phylogenetic position of the new species within the family Macrobiotidae. The new species has a microplacoid that placed it within Parmacrobiotus richtersi group and exhibit richtersi-type eggs having processes terminated with cap-like structures. Paramacrobiotus gadabouti sp. nov. is most similar to Pam. alekseevi, Pam. filipi and Pam. garynahi, but differs from them mainly in details of egg morphology and morphometrics. Unlike other species from this group, which were confirmed as bisexual and showed limited distribution, Paramacrobiotus gadabouti sp. nov. is yet another parthenogenetic species with a wide distribution, demonstrating that at least some tardigrades confirm to the hypothesis of 'everything is everywhere'., (© 2023. The Author(s).)

Tardigrada is an invertebrate phylum that often constitutes a dominant micrometazoan group on glaciers worldwide. We investigated tardigrades residing in surface ice above the equilibrium line altitude (ELA) on three temperate glaciers of New Zealand's Southern Alps. Morphological, morphometric and multilocus DNA analyses (CO1, 18S rRNA, 28S rRNA, ITS-2) revealed two new genera comprising four species, of which two are formally described here: Kopakaius gen. nov. nicolae sp. nov. and Kararehius gen. nov. gregorii sp. nov. The former is represented by three genetically distinct phyletic lineages akin to species. According to CO1, Kopakaius gen. nov. nicolae sp. nov. inhabits Whataroa Glacier only while the remaining two Kopakaius species occur on Fox and Franz Joseph Glaciers, suggesting low dispersal capabilities. Although morphological characteristics of the new genera could indicate affinity with the subfamily Itaquasconinae, phylogenetic analysis placed them confidently in the subfamily Diphasconinae. Kopakaius gen. nov. lack placoids in the pharynx similar with some Itaquasconinae, whereas dark pigmentation and claw shape aligns them with the glacier-obligate genus, Cryobiotus (subfamily Hypsibiinae), which is an example of parallel evolution. The second genus, Kararehius gen nov. could be classified as Adropion-like (subfamily Itaquasconinae), but differs greatly by genetics (placed in the subfamily Diphasconinae) as well as morphology (e.g., lack of septulum), exemplify deep stasis in Hypsibiidae. Our results suggest that glacier fragmentation during the Pleistocene triggered tardigrade speciation, making it a suitable model for studies on allopatric divergence in glacier meiofauna., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

This work presents two new Mesobiotus species from the Republic of South Africa, formally described using integrative analyses. Specimens of the new species are examined in terms of morphology and morphometry under a contrast phase light microscope (PCM) and scanning electron microscope (SEM). For both new species, genetic data in the form of DNA sequences of commonly used molecular markers are also provided (18S rRNA, 28S rRNA, COI , ITS-2). Furthermore, such genetic data are also provided for the first time for Mesobiotus peterseni (Maucci, 1991) from Greenland. The study also presents a multilocus molecular phylogeny of the genus and an elaborated discussion on the taxa groupings and species composition. This results in the ratification of three informal morpho-groups in order to ease and improve communication in further taxonomic studies on the genus. Finally, an updated key to all valid nominal Mesobiotus taxa (71 species) is provided to enhance species identification in this morphologically diverse group of limno-terrestrial tardigrades.

A new tardigrade species of the genus Macrobiotus C.A.S. Schultze, 1834 from Kyrgyzstan, is described and illustrated in this paper. Macrobiotus rebecchii sp. nov. is a hermaphroditic and limnoterrestrial species found in a moss growing on a rock in Toluk village. Specimens of the new species were examined for its morphological details using contrast phase light microscope (PCM) and scanning electron microscope (SEM). Genetic data in the form of DNA sequences of commonly used molecular markers were also obtained (18S rRNA, COI). Phenotypically the new species is most similar to Macrobiotus joannae Pilato & Binda, 1983, Macrobiotus punctillus Pilato, Binda & Azzaro, 1990, and Macrobiotus hannae Nowak & Stec, 2018, but can be easily differentiated from all of them by its body granulation pattern.

Animals that colonize soil show specific adaptations to soil. Compared to closely related species living on the surface, the limbs of soil-dwelling animals are often shortened, reduced, or absent to allow a less restricted passage through cavities between soil particles. This pattern of limb reduction has also been observed in tardigrades, where multiple lineages that colonized the below-ground habitat show independent reduction and/or loss of legs and claws. In the tardigrade superfamily Macrobiotoidea, leg and claw reductions are a common trait found in the Macrobiotus pseudohufelandi complex. This rarely found species complex currently contains four nominal taxa. Here we describe, with the use of integrative taxonomy, Macrobiotus naginae sp. nov., a new species in the Macrobiotus pseudohufelandi complex from inland sand dunes in Finland. We also provide a dichotomous key to the Macrobiotus pseudohufelandi complex to assist with their identification in future studies.

Revisions and redescriptions of taxa described in the past and that are now categorized as insufficiently diagnosed often play a crucial role in making further progress in modern taxonomy in many groups of organisms. Here we revised an enigmatic tardigrade species Tenuibiotus hyperonyx (Maucci, 1983) based on the newly discovered topotypic population from the Italian Alps. We performed an integrative analysis of morphological and genetic data in order to present an upgraded species description and elucidate its phylogenetic position. Our results enabled us to confidently place T. hyperonyx within the family Richtersiusidae, as a member of the genus Diaforobiotus . This change, together with a re-assessment of microphotographs of the Tenuibiotus willardi (Pilato, 1977) and Tenuibiotus bozhkae Pilato, Kiosya, Lisi, Inshina & Biserov, 2011 types, led to the discussion on species composition with narrative taxa amendments for the taxonomic parties involved in the proposed alteration.

The family Macrobiotidae is one of the most speciose and diverse groups among tardigrades. Although there have been attempts to reconstruct the phylogeny of this family, the evolutionary relationships within Macrobiotidae are only superficially determined as available genetic data cover only a small fraction of this vast group. Here, we present the first extensive molecular phylogeny of the family based on four molecular markers (18S rRNA, 28Sr RNA, ITS-2 and COI) associated with detailed morphological data for the majority of taxa. The phylogenetic analysis includes nearly two hundred sequences representing more than sixty species, including sixteen taxa that have never been sequenced and/or analysed phylogenetically before. Our results recovered a new monophyletic group, comprising Macrobiotus spectabilis Thulin, 1928 and Macrobiotus grandis Richters, 1911, for which we erect a new genus, Sisubiotusgen. nov., to accommodate its evolutionary distinctiveness. The largest, so far, dataset for the family Macrobiotidae showed that the genus Xerobiotus is nested within the clade representing the genus Macrobiotus deeper than it was earlier assumed, therefore we propose to suppress Xerobiotus and transfer its species to Macrobiotus. Moreover, mapping key morphological traits onto macrobiotid phylogeny exposed complex evolution of phenotypes within the Macrobiotus hufelandi group, i.e. Macrobiotus s.s. Finally, our findings enabled a detailed revision and discussion on species compositions of the most ubiquitous tardigrade genera, species groups and species complexes, which resulted in changes of taxonomic statuses of a number of macrobiotid species. All this contributes to the reconstruction of the morphological evolution within Macrobiotidae., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The honeybee continues to be developed as a model species in many research areas, including studies related to the effects of alcohol. Here, we investigate whether workers display one of the key features of alcoholism, namely withdrawal symptoms. We show that workers fed for a prolonged time on food spiked with ethanol, after discontinuation of access to such food, exhibited a marked increase in the consumption of ethanol and a slight increase in mortality. We additionally show that withdrawal symptoms do not include an increase in appetitiveness of ethanol diluted in water. Our results demonstrate that workers can develop alcohol dependence, which might be especially important in the natural setting of repeated exposure to ethanol in floral nectar and for their potential as a model of alcohol addiction.

The taxonomy of many groups of meiofauna is challenging due to their low number of diagnostic morphological characters and their small body size. Therefore, with the advent of molecular techniques that provide a new source of traits, many cryptic species have started to be discovered. Tardigrades are not an exception, and many once thought to be cosmopolitan taxa are being found to be complexes of phenotypically similar species. Macrobiotus pallarii Maucci, 1954 was originally described in South Italy and has been subsequently recorded in Europe, America, and Asia. This allegedly wide geographic range suggests that multiple species may be hidden under this name. Moreover, recently, genetic evidence to support this was put forward, and the Macrobiotus pallarii complex has been proposed to accommodate putative species related to M. pallarii. Here, we describe three new pseudocryptic species based on populations that would have been all classified as Macrobiotus pallarii if molecular methods were not employed. Using an integrative taxonomy approach, we analyzed animals and eggs from the topotypic population of Macrobiotus pallarii, together with four other populations of the complex. We recovered four distinct phylogenetic lineages that, despite the overlap of morphometric traits, can be separated phenotypically by subtle but discrete morphological characters. One lineage corresponds to Macrobiotus pallarii, whereas the other three are newly described as Macrobiotus margoae Stec, Vecchi & Bartels, sp. nov. from the USA, Macrobiotus ripperi Stec, Vecchi & Michalczyk, sp. nov. from Poland and Finland, and Macrobiotus pseudopallarii Stec, Vecchi & Michalczyk, sp. nov. from Montenegro. To facilitate species identification, we provide a dichotomous key for species of the M. pallarii complex. Delimitation of these pseudocryptic taxa highlights the need for an integrative approach to uncover the phylum's diversity in full.

In a moss and lichen sample collected on the Polish coast, a new population of Macrobiotus sottilei was found. Given that the original description of M. sottilei was based solely on the morphology observed under light microscopy and measurements of a few individuals, we provide, by means of integrative taxonomy, a revisional note on this species. We present a comprehensive set of morphometric and morphological data from light and scanning electron microscopy analysis together with nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragments. We also provide the same set of DNA sequences for Macrobiotus glebkai from a population recently found in Eastern Ukraine and elucidate the phylogenetic position of these two taxa within the family Macrobiotidae. Moreover, the terminology of cuticular bars in macrobiotid legs, and pillars within the egg chorion ornamentation in the Macrobiotus hufelandi morphogroup, are also discussed.

Some species of slugs belonging to the genus Deroceras are invasive and cause severe agricultural damage. Despite extensive knowledge about their invasiveness, data on the molecular differentiation of these morphologically similar species are lacking. Here we present a molecular approach to identifying three closely related species of the genus Deroceras - D. agreste (L., 1758), D. reticulatum (O. F. Müller, 1774) and D. turcicum (Simroth, 1894) (Gastropoda: Eupulmonata: Agriolimacidae)-based on sequences of multiple molecular markers: cytochrome c oxidase subunit I ( COI ), cytochrome b (cyt- b ), internal transcribed spacer 2 (ITS-2) and 28S ribosomal RNA (28S rRNA). We also provide detailed photomicrographs of the penis and penial gland of the three species, as it is the latter that holds the most important phenotypic characters for distinguishing between these taxa. Since identification of the studied species based solely on morphology is considered challenging, contributing a means of molecular differentiation will aid further ecological and biodiversity surveys of these important pests.

The family Richtersiidae, although established recently with the use of phylogenetic methods, was considered potentially paraphyletic at the time of its erection. Until now, the family comprised four genera, Richtersius, Diaforobiotus, Adorybiotus and a newly erected genus Crenubiotus. However, the genetic characterisation for the latter two genera was very limited or absent. To address concerns about the phylogenetic affinity of these two genera, we present a multilocus phylogeny of the families Richtersiidae and Murrayidae based on four molecular markers (18S rRNA, 28S rRNA, ITS-2 and COI). Our results show a distinct evolutionary lineage composed of Adorybiotus and Crenubiotus, which is sister to Murrayidae. In order to accommodate the phylogenetic and morphological distinctiveness of this lineage, we erect a new family, Adorybiotidae fam. nov. The new taxon differs morphologically from other families in the superfamily Macrobiotoidea by a unique combination of traits: (1) the presence of tubercles/cushions with aggregations of microgranules on their surfaces present on all legs and on the dorso-caudal cuticle, (2) a system of internal septa in claws, and (3) buccal apparatus morphology. Moreover, in order to stabilise the taxonomy and nomenclature in the genus Crenubiotus, we redescribe its type species, Crenubiotus crenulatus, by means of integrative taxonomy and designate a new neotype based on a population from the original terra typica.

The designation of a neotype for Macrobiotus coronifer Richters, 1903 (now the type species of the genus Richtersius Pilato Binda, 1989) by Maucci Ramazzotti (1981) with type locality Bodø in Norway is shown to be invalid as it does not comply with the International Code of Zoological Nomenclature (Article 75.3.4). Furthermore, the specimen selected by Maucci Ramazzotti (1981) is not from the original type locality, and the superficial and outdated documentation prevent a reliable identification of the species. A Code-compliant neotype is therefore designated. The new neotype was collected from the original locus typicus in Svalbard and described with standard light microscopy, detailed scanning electron microscopy imaging, DNA barcodes and a transcriptome, which makes it ideally suited for stabilising the taxonomy and nomenclature of Richtersius coronifer (Richters, 1903).

Research on the behavioral responses of animals to extreme weather events, such as heat wave, is lacking even though their frequency and intensity in nature are increasing. Here, we investigated the behavioral response to a simulated heat wave in two species of antlions (Neuroptera: Myrmeleontidae). These insects spend the majority of their lives as larvae and live in sandy areas suitable for a trap-building hunting strategy. We used larvae of Myrmeleon bore and Euroleon nostras , which are characterized by different microhabitat preferences-sunlit in the case of M. bore and shaded in the case of E. nostras . Larvae were exposed to fluctuating temperatures (40 °C for 10 h daily and 25 °C for the remaining time) or a constant temperature (25 °C) for an entire week. We found increased mortality of larvae under heat. We detected a reduction in the hunting activity of larvae under heat, which corresponded to changes in the body mass of individuals. Furthermore, we found long-term consequences of the simulated heat wave, as it prolonged the time larvae needed to molt. These effects were pronounced in the case of E. nostras but did not occur or were less pronounced in the case of M. bore , suggesting that microhabitat-specific selective pressures dictate how well antlions handle heat waves. We, thus, present results demonstrating the connection between behavior and the subsequent changes to fitness-relevant traits in the context of a simulated heat wave. These results illustrate how even closely related species may react differently to the same event., (© The Author(s) 2020. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology.)

In this paper we describe two new tardigrade species, one representing the Macrobiotus hufelandi complex and the other from the Paramacrobiotus richtersi complex. The descriptions are based on a detailed morphological examination under light and scanning electron microscopy and analysis of four genetic markers (18S rRNA, 28S rRNA, ITS-2 and COI ). Macrobiotus crustulus sp. nov. from French Guiana is the most similar to Macrobiotus martini Bartels, Pilato, Lisi and Nelson, 2009, Macrobiotus santoroi Pilato and D'Urso, 1976, but differs from them mainly by having the lissostomus type of the oral cavity armature (teeth not visible under light microscopy) and well-developed, convex terminal discs of egg processes covered with evident granulation. Paramacrobiotus filipi sp. nov. from the Malaysian part of Borneo is the most similar to Paramacrobiotus alekseevi (Tumanov, 2005), but differs from it primarily by the presence of body granulation visible under light microscopy as well as sculptured and porous areoles around egg processes.

Richtersius coronifer , the nominal species for the family Richtersiidae and a popular laboratory model, exemplifies a common problem in modern tardigrade taxonomy. Despite undeniable progress in the field, many old and incomplete descriptions of taxa hinder both species delimitation and the estimation of species diversity and distribution. Although for over a century this species has been recorded throughout the world, recent research indicates that records to date are likely to represent a species complex rather than a single cosmopolitan species. However, in order to recognise and name species diversity within the complex, an integrative redescription of the nominal species is first needed. Here, we describe an R. coronifer population collected from Spitsbergen, i.e., one of the two localities mentioned in the original description, with detailed morphological and morphometric data associated with standard DNA sequences of four standard genetic markers (18S rRNA, 28S rRNA, ITS-2, and COI) and supported by transcriptome sequencing. We propose replacement of the neotype designated in 1981 by Maucci and Ramazzotti, as it is impossible to verify whether the existing neotype is conspecific with specimens studied by Richters in 1903 and 1904. Finally, using newly obtained cytochrome c oxidase subunit I (COI) sequences of populations from Spitsbergen, Italy, Poland, and Greece together with sequences deposited in GenBank (China, Greenland, Italy, Mongolia), we performed genetic species delimitation, which indicated seven distinct potential species within the genus Richtersius , in addition to the nominal taxon. This study marks a starting point for further research on the taxonomy of and species diversity within the genus. Moreover, this work has the potential to be the first tardigrade redescription to provide both genetic barcodes and a transcriptome of the species in question., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

The genus Echiniscus C.A.S. Schultze, 1840, one of the earliest established and speciose tardigrade genera, has been hypothesized to be polyphyletic. Moreover, the presence or absence of trunk appendages, the type of cuticular sculpturing and body colour have been argued to hold taxonomic significance at the genus level in Echiniscus-line taxa. Here, by combining morphological and genetic analyses, we demonstrate that the so-called "arctomys group", i.e. Echiniscus spp. lacking trunk appendages, comprises numerous separate evolutionary lineages within the family Echiniscidae. As a result, we erect five new echiniscid genera: Barbariagen. nov., presumably of Neotropical (Gondwanan) origin, previously classified as the Echiniscus bigranulatus group; the pantropical and subtropical Kristenseniscusgen. nov. (the tessellatus group), characterized by a peculiar subdivision of dorsal plates; Claxtoniagen. nov. (the wendti group), with large and evident endocuticular pillars in the form of polygons; Nebularmisgen. nov. (the reticulatus group), with an elusive dorsal sculpturing; and Viridiscusgen. nov. (the viridis group), with body colour ranging from light green through brownish to even almost black. Additionally, we briefly address appendaged Echiniscus s.s. and divide the genus into several groups based on dorsal plate sculpturing and suggest that these could also represent separate supraspecific entities., (© The Willi Hennig Society 2019.)

A new species of the Mesobiotus harmsworthi group is described from Datanla Waterfall amusement park in Lâm Đồng Province, Vietnam. An integrative taxonomy approach was applied, by combining morphological and morphometric analyses using light contract microscopy (LCM)) and scanning electron microscopy (SEM) with genetic analysis (18S rRNA, 28S rRNA, ITS-2 and COI markers), to cross-verify the status of the newly discovered population as a new species. By the presence of fully developed areolation around egg processes bases and well developed oral cavity armature (OCA), with all three bands of teeth visible under (LCM), the new species, Mesobiotus datanlanicus sp. nov., is most similar to seven taxa from the harmsworthi complex: M. barbarae (Kaczmarek, Michalczyk Degma, 2007), M. hieronimi (Pilato Claxton, 1988), M. insanis Mapalo, Stec, Mirano-Bascos Michalczyk, 2017, M. neuquensis (Rossi, Claps Ardohain, 2009), M. nuragicus (Pilato Sperlinga, 1975), M. pseudoliviae (Pilato Binda, 1996), M. pseudonuragicus (Pilato, Binda, Lisi, 2004), but differs from them mainly by some specific characters of the egg and adult morphology as well as by morphometrics.

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., Competing Interests: The authors have declared that no competing interests exist.

The family Echiniscidae comprises limno-terrestrial heterotardigrades with a strongly sclerotised dorsum, typically covered with plates. Among other members of the Echiniscus evolutionary line, the genus Testechiniscus Kristensen, 1987 stands out with well-developed ventral armature and polygonal sculpturing of the dorsal plates. It has alleged bipolar distribution (with satellite alpine records in the Holarctic). Thanks to fresh material from terra typica (Svalbard), we integratively redescribe (i.e. using light and electron microscopy imaging, morphometry, and molecular methods) the nominal species for the genus, Testechiniscus spitsbergensis (Scourfield, 1897). A comparison of the neotype series with a number of Holarctic records revealed morphological variability suggesting that the species may encompass several taxa, which, most likely, will be possible to delineate primarily with molecular tools. Moreover, based on material from Simien Mountains (Northern Ethiopia), we describe a new sibling subspecies, Testechiniscus spitsbergensis tropicalis ssp. nov. Extensive morphometric datasets are provided for the genus members for the first time. A new generic definition is proposed, embracing the two subspecies of T. spitsbergensis and T. laterculus (Schuster et al., 1980), but excluding two circum-Antarctic species, T. macronyx (Richters, 1907) and T. meridionalis (Murray, 1906). The later species are likely to be erected in the future as separate genera, and their autapomorphies are described here. In the light of our findings, the genus          Testechiniscus should be recognised as a native element of the Northern Hemisphere, with mainly circum-Arctic distribution and additional, insular alpine records from the Nearctic, Palearctic and Eastern Afrotropic.

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.

In this paper we describe Macrobiotus papei, a new species of the Macrobiotus hufelandi complex from the Udzungwa Mountains National Park in Tanzania. Our research included the traditional morphology-based taxonomic analysis, supported by morphometrics, light and scanning electron microscopy imaging, as well as analysis of nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) marker. The molecular sequences provided a more accurate description and independent verification of the taxonomic status of M. papei sp. nov. Such integrative approach requires a considerable number of individuals and eggs, which we obtained by establishing a laboratory culture of the new species. Our analyses revealed that M. papei sp. nov., by having the flexible filaments on the terminal discs of the egg processes, is most similar to Macrobiotus paulinae Stec et al., 2015 from Africa and Macrobiotus polypiformis Roszkowska et al., 2017 from South America. However, the new species can be easily distinguished from both these species by patches of cuticular granulation on the external and internal surface of legs I-III (only external patches present in in M. paulinae and M. polypiformis), two separate lateral transversal ridges with a rounded median tooth between instead of a single thin ventral transverse ridge in the third band of teeth in the oral cavity, smooth terminal discs and filaments on the egg processes (terminal discs and filaments covered by aggregations of small microgranules in M. paulinae and M. polypiformis), and by some morphometric characters of both adults and eggs.

A laboratory strain identified as "Hypsibius dujardini" is one of the best studied tardigrade strains: it is widely used as a model organism in a variety of research projects, ranging from developmental and evolutionary biology through physiology and anatomy to astrobiology. Hypsibius dujardini, originally described from the Île-de-France by Doyère in the first half of the 19th century, is now the nominal species for the superfamily Hypsibioidea. The species was traditionally considered cosmopolitan despite the fact that insufficient, old and sometimes contradictory descriptions and records prevented adequate delineations of similar Hypsibius species. As a consequence, H. dujardini appeared to occur globally, from Norway to Samoa. In this paper, we provide the first integrated taxonomic redescription of H. dujardini. In addition to classic imaging by light microscopy and a comprehensive morphometric dataset, we present scanning electron photomicrographs, and DNA sequences for three nuclear markers (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial marker (COI) that are characterised by various mutation rates. The results of our study reveal that a commercially available strain that is maintained in many laboratories throughout the world, and assumed to represent H. dujardini sensu stricto, represents, in fact, a new species: H. exemplaris sp. nov. Redescribing the nominal taxon for Hypsibiidae, we also redefine the family and amend the definitions of the subfamily Hypsibiinae and the genus Hypsibius. Moreover, we transfer H. arcticus (Murray, 1907) and Hypsibius conifer Mihelčič, 1938 to the genus Ramazzottius since the species exhibit claws and eggs of the Ramazzottius type. Finally, we designate H. fuhrmanni as subjectively invalid because the extremely poor description precludes identifying neotype material.

Tardigrade research in Japan dates back over 100 years, and to date, 167 species of this ecdysozoan phylum have been reported from the country. Of these species, the Macrobiotus hufelandi complex has been represented only by the nominal taxon of this group, Macrobiotus hufelandi. In this article, a new species of the hufelandi group from Japan, Macrobiotus shonaicus sp. nov., is described using integrative taxonomy. In addition to the detailed morphological and morphometric data, obtained using phase contrast light microscopy (PCM) and scanning electron microscopy (SEM), we provide DNA sequences of four molecular markers (both nuclear and mitochondrial). The new species belongs to the persimilis subgroup and is most similar to M. anemone from USA, M. naskreckii from Mozambique, and M. patagonicus from Argentina, but it can be easily distinguished from these species by the presence of thin flexible filaments on terminal discs of the egg process. By the latter character, the new species is most similar to M. paulinae and M. polypiformis, but it can be easily distinguished from them by having a solid egg surface between egg processes (i.e., without pores or reticulum). A phylogenetic analysis of available DNA sequences of the COI marker for the hufelandi group revealed that the new species clusters with the two other species that exhibit filaments on egg process discs (M. paulinae and M. polypiformis) and with two species that have entire egg processes modified into filaments (M. kristenseni and M. scoticus). All five species form a clade distinct from all other sequenced species of the hufelandi group with typical mushroom- or inverted goblet-shaped egg processes, which may suggest that the ancestor of the five species with atypical egg processes had a mutation allowing derivations from the mushroom or inverted chalice-like shape of egg processes.

Nearly a half of known eutardigrade species lay ornamented eggs. The ornamentation is thought to provide attachment of the egg to the substrate and protection for the developing embryo, but from the taxonomic point of view chorion morphology may also provide key characters for species differentiation and identification, especially between closely related taxa. Nonetheless, despite the evolutionary and taxonomic importance of the egg shell, the determinants of its morphology are very poorly, if at all, understood. Here, we combine morphological, molecular and experimental approaches in an attempt to separate the genetic and environmental factors that shape egg chorion morphology in Ramazzottius subanomalus (Biserov, 1985). Our integrative study, based on a population of R. subanomalus isolated from a single moss sample, revealed (1) remarkable variation in egg shell morphology, but (2) relatively little variation in animal morphometric traits, and (3) genetic differentiation, expressed as two ITS-2 haplotypes, but no parallel polymorphism in COI. Although animals did not differ morphometrically between the haplotypes, eggs laid by haplotype 1 and 2 females exhibited highly statistically significant differences in all measured traits. The study demonstrates, for the first time, a correlation between phenotypic and genetic variability within a tardigrade species. The revealed congruence between genetic and morphological traits might be viewed as an example of incipient speciation that illustrates early evolutionary steps leading to species complexes that differ primarily in terms of egg shell morphology. Moreover, our data confirm the value of the ITS-2 fragment in distinguishing very closely related tardigrade lineages.

The limnoterrestrial tardigrade fauna of the Philippines is completely unknown. In this paper, we describe the first ever limnoterrestrial water bear species from this southeast Asian country, Mesobiotus philippinicus sp. nov., found in a moss sample collected in Quezon City. Apart from morphometrics and imaging in light microscopy, we also analysed the new species under scanning electron microscope and sequenced four DNA markers differing in mutation rates, three nuclear (18S rRNA, 28S rRNA, and ITS-2) and one mitochondrial (COI). This allowed not only a detailed description but also provided barcodes to aid future species identification. The new species belongs to the harmsworthi group and is most similar to M. diffusus (Binda & Pilato, 1987), M. pseudocoronatus (Pilato et al., 2006), M. montanus (Murray, 1910) and M. mottai (Binda & Pilato, 1994), but differs from these species by whorled egg processes and dimensions of some morphometric traits. The 28S rRNA, ITS-2 and COI sequences presented in this paper are the first published DNA sequences for the genus Mesobiotus.

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.

In this paper we describe Macrobiotus paulinae, a new species of the hufelandi group from the Kenyan highlands. In addition to the traditional taxonomic description, aided with morphometrics as well as light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragment of the new species. The sequences allowed not only a more accurate description but also provided an independent verification of the taxonomic status of Ma. paulinae sp. nov. Such integrative approach requires a considerable number of individuals and eggs, which we have partially subsidised by employing an in vitro culture of the new species. Our analyses revealed that Ma. paulinae sp. nov. is most similar to Macrobiotus madegassus Maucci, 1993 and Macrobiotus modestus Pilato & Lisi, 2009, however it differs from these species, as well as from all other known species of the hufelandi group, by the presence of seven paired dorso-lateral patches of cuticular granulation and the presence of chorionic filaments growing out of terminal discs of egg processes. Macrobiotus paulinae sp. nov. is an example of a species with a miniaturised buccal apparatus (i.e. with reduced peribuccal lamellae and oral cavity armature, and stylet supports inserted on the buccal tube more anteriorly than in typical Ma. hufelandi group species), and it therefore resembles two recently described two-macroplacoided Minibiotus species: Mi. acadianus Meyer & Domingue, 2011 and Mi. julianae Meyer, 2012. The re-examination of the type material for these two species confirmed that they are equipped with peribuccal lamellae and therefore we transfer them to the genus Macrobiotus, specifically to the hufelandi group.

The acute toxicity of ammonia on Thulinius ruffoi (Bertolani, 1981), a eutardigrade isolated from a small waste water treatment plant (WWTP) in Poland, was estimated. Our results show that no active individuals survived a 24 h exposure to solutions equal to or higher than 125 mg/L of total ammonia nitrogen (NH3-N + NH4 (+)-N), which, under the conditions in our experiment, was equivalent to 1.17 mg/L of un-ionised ammonia (NH3). The LC50 concentration of total ammonia nitrogen was equal to 52 mg/L (or 0.65 mg/L un-ionised ammonia). Given that the norms for the concentration of ammonia in treated waters leaving WWTPs are usually several times lower than the LC50 for T. ruffoi, this species does not seem to be a good bioindicator candidate for WWTPs. In this paper we also note that various ecotoxicological studies use different methodological approaches and we suggest that a more uniform methodology may aid interspecific comparisons of LC50 values.