Pinpointing the microbiota of tardigrades: What is really there?

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.

Expanding Acutuncus: Phylogenetics and morphological analyses reveal a considerably wider distribution for this tardigrade genus.

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.

Two new tardigrade genera from New Zealand's Southern Alp glaciers display morphological stasis and parallel evolution.

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.

New multilocus phylogeny reorganises the family Macrobiotidae (Eutardigrada) and unveils complex morphological evolution of the Macrobiotus hufelandi group.

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.

Discontinued alcohol consumption elicits withdrawal symptoms in honeybees.

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.

Untangling the Echiniscus Gordian knot: paraphyly of the "arctomys group" (Heterotardigrada: Echiniscidae).

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.

Tolerance to Ammonia of Thulinius ruffoi (Bertolani, 1981), a Tardigrade Isolated from a Sewage Treatment Plant.

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.

Occasional and constant exposure to dietary ethanol shortens the lifespan of worker honey bees.

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.

Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae).

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'.

Macrobiotus rebecchii sp. nov.: A New Limno-Terrestrial and Hermaphroditic Tardigrade from Kyrgyzstan.

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.

An Integrative Description of Two New Mesobiotus Species (Tardigrada: Eutardigrada: Macrobiotidae) with Updated Genus Phylogeny.

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.

Reaching the Monophyly: Re-Evaluation of the Enigmatic Species Tenuibiotus hyperonyx (Maucci, 1983) and the Genus Tenuibiotus (Eutardigrada).

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.

Macrobiotus naginae sp. nov., a New Xerophilous Tardigrade Species from Rokua Sand Dunes (Finland).

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.

Phylogenetic position of two Macrobiotus species with a revisional note on Macrobiotus sottilei Pilato, Kiosya, Lisi amp; Sabella, 2012 (Tardigrada: Eutardigrada: Macrobiotidae).

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.

Integrative taxonomy resolves species identities within the Macrobiotus pallarii complex (Eutardigrada: Macrobiotidae).

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.

Macrobiotus coronifer Richters, 1903 (type species for Richtersius Pilato amp; Binda, 1989): designating a new neotype from the original type locality described within the integrative taxonomy framework.

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).

Molecular Approach to Identifying Three Closely Related Slug Species of the genus Deroceras (Gastropoda: Eupulmonata: Agriolimacidae).

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.

Heat wave effects on the behavior and life-history traits of sedentary antlions.

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.

Integrative Descriptions of Two New Macrobiotidae Species (Tardigrada: Eutardigrada: Macrobiotoidea) from French Guiana and Malaysian Borneo.

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.

Resolving the systematics of Richtersiidae by multilocus phylogeny and an integrative redescription of the nominal species for the genus Crenubiotus (Tardigrada).

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.