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.

First evidence of sex-specific responses to chemical cues in tardigrade mate searching behaviour.

Chemical cues are widely used in intraspecific and interspecific communication, either as substances deposited in the substrate or as molecules diffused in water or air. In tardigrades, an emerging microscopic study system, chemical communication and its role in reproduction are poorly known. Here, we assessed sex differences in the detection of (a) short-range diffusing signals and (b) deposited cue trails during the mate-searching behaviour of freely moving virgin male and female Macrobiotus polonicus. We tracked individual behaviour (a) in simultaneous double-choice chambers, where live conspecifics of each sex were presented in water and (b) of freely moving pairs on agar without water. We found that males, but not females, preferentially associated with opposite-sex individuals in trials conducted in water. In contrast, neither sex detected nor followed cues deposited on agar. In conclusion, our study suggests that mate discrimination and approach are male-specific traits and are limited to waterborne chemical cues. These results support the existence of Darwinian sex roles in pre-mating behaviour in an animal group with virtually non-existing sex differences in morphology or ecology.

Fixation offset decreases pupillary inhibition of return.

Inhibition of return (IOR) is reflected as a slower manual or saccadic response to a cued rather than an uncued target (manual IOR and saccadic IOR, respectively), and as a pupillary dilation when a bright, relative to a dark side of a display is cued (pupillary IOR). The aim of this study was to investigate the relation between an IOR and oculomotor system. According to the predominant view, only the saccadic IOR is strictly related to the visuomotor process, and the manual and pupillary IORs depend on non-motor factors (e.g., short-term visual depression). Alternatively, the after-effect of the covert-orienting hypothesis postulates that IOR is strictly related to the oculomotor system. As fixation offset affects oculomotor processes, this study investigated whether fixation offset also affects pupillary and manual IORs. The results show that fixation offset decreased IOR in pupillary but not manual responses, and provides support for the hypothesis that at least the pupillary IOR is tightly linked to eye movement preparation.

"Everything is not everywhere": Time-calibrated phylogeography of the genus Milnesium (Tardigrada).

There is ample evidence that macroscopic animals form geographic clusters termed as zoogeographic realms, whereas distributions of species of microscopic animals are still poorly understood. The common view has been that micrometazoans, thanks to their putatively excellent dispersal abilities, are subject to the "Everything is everywhere but environment selects" hypothesis (EiE). One of such groups, <1 mm in length, are limnoterrestrial water bears (Tardigrada), which can additionally enter cryptobiosis that should further enhance their potential for long distance dispersion (e.g., by wind). However, an increasing number of studies, including the most recent phylogeny of the eutardigrade genus Milnesium, seem to question the general applicability of the EiE hypothesis to tardigrade species. Nevertheless, all Milnesium phylogenies published to date were based on a limited number of populations, which are likely to falsely suggest limited geographic ranges. Thus, in order to test the EiE hypothesis more confidently, we considerably enlarged the Milnesium data set both taxonomically and geographically, and analysed it in tandem with climate type and reproductive mode. Additionally, we time-calibrated our phylogeny to align it with major geological events. Our results show that, although cases of long distance dispersal are present, they seem to be rare and mostly ancient. Overall, Milnesium species are restricted to single zoogeographic realms, which suggests that these tardigrades have limited dispersal abilities. Finally, our results also suggest that the breakdown of Gondwana may have influenced the evolutionary history of Milnesium. In conclusion, phylogenetic relationships within the genus seem to be determined mainly by paleogeography.

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.

Sexual selection protects against extinction.

Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through 'genic capture' could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress.

Preparation of saccade sequences and eye programming affect endogenous covert attention.

Endogenous attention can be allocated in parallel to at least two saccade target locations of a planned sequence, but attentional resources are larger in the location of the first than the second saccade. The meridian effect that is observed in endogenous attention can be explained by eye programming, but it is not known how eye-movement preparation and eye programming can together affect endogenous attention during sequential saccades. We used a double-task paradigm to investigate this issue. In two experiments, we confirmed the relation between the preparation of sequential saccades and attentional selection and also showed that eye programming could eliminate deterioration of attentional resources in the second saccade location. The finding of the meridian effect in both the saccadic task and the target discrimination task additionally indicates the important role of eye programming in endogenous attention. The results were discussed in terms of the Premotor Theory of Attention.

Mating patterns influence vulnerability to the extinction vortex.

Earth's biodiversity is undergoing mass extinction due to anthropogenic compounding of environmental, demographic and genetic stresses. These different stresses can trap populations within a reinforcing feedback loop known as the extinction vortex, in which synergistic pressures build upon one another through time, driving down population viability. Sexual selection, the widespread evolutionary force arising from competition, choice and reproductive variance within animal mating patterns could have vital consequences for population viability and the extinction vortex: (a) if sexual selection reinforces natural selection to fix 'good genes' and purge 'bad genes', then mating patterns encouraging competition and choice may help protect populations from extinction; (b) by contrast, if mating patterns create load through evolutionary or ecological conflict, then population viability could be further reduced by sexual selection. We test between these opposing theories using replicate populations of the model insect Tribolium castaneum exposed to over 10 years of experimental evolution under monogamous versus polyandrous mating patterns. After a 95-generation history of divergence in sexual selection, we compared fitness and extinction of monogamous versus polyandrous populations through an experimental extinction vortex comprising 15 generations of cycling environmental and genetic stresses. Results showed that lineages from monogamous evolutionary backgrounds, with limited opportunities for sexual selection, showed rapid declines in fitness and complete extinction through the vortex. By contrast, fitness of populations from the history of polyandry, with stronger opportunities for sexual selection, declined slowly, with 60% of populations surviving by the study end. The three vortex stresses of (a) nutritional deprivation, (b) thermal stress and (c) genetic bottlenecking had similar impacts on fitness declines and extinction risk, with an overall sigmoid decline in survival through time. We therefore reveal sexual selection as an important force behind lineages facing extinction threats, identifying the relevance of natural mating patterns for conservation management.

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.

The gap effect reduces both manual and saccadic inhibition of return (IOR).

Inhibition of return (IOR) is the effect of slower responses to validly than invalidly cued targets. The discovery of IOR raised controversy as to whether it has two "flavors", i.e., attentional/perceptual and motoric, or whether it is a homogeneous visual-motor phenomenon that should be understood in terms of the preparation of different effectors (mainly eye movement). Since manipulation of fixation offset (0 and 200 ms gap) is believed to affect the latency of saccades, we measured its influence on saccadic and manual IOR with a simple keypress response when eye movements were forbidden. In the two experiments which we carried out, the fixation offset decreased IOR in both the saccadic and the manual conditions. The results suggest the limitations of the attentional hypothesis, which assumes that manual IOR is independent of the motoric component; they are also in line with the tenets of the oculomotor hypothesis of IOR.

Fitness consequences of threshold trait expression subject to environmental cues.

Most cases of alternative reproductive tactics (ARTs) are thought to represent conditional strategies, whereby high-status males express highly competitive phenotypes, whereas males below a certain status threshold resort to sneaky tactics. The underlying evolutionarily stable strategy (ESS) model assumes that males of high competitive ability achieve higher fitness when expressing the territorial phenotype, whereas the less competitive males are more fit as sneakers, caused by fitness functions for the ARTs having different slopes and intersecting at a threshold value of competitive ability. The model, however, is notoriously difficult to test as it requires access to low-status territorials and high-status sneakers, that rarely occur in nature. Here, we test the conditional ESS in the androdimorphic acarid mite Sancassania berlesei, where large males tend to develop into an armoured, aggressive 'fighter' morph, while small males become unarmoured, non-aggressive 'scramblers'. In addition to body size, male morph is affected by pheromones produced by big populations, with fighters being suppressed in dense colonies. By manipulating pheromone concentration, we obtained high-status scramblers and low-status fighters. We also estimated status- and size-dependent fitness functions for male morphs across a range of population sizes. Fighters had the highest fitness in small populations and their fitness declined with increasing density, whereas the reverse was true for scramblers, providing support for condition-dependent ESS with respect to demography. However, whereas male fitness increased with body size, the fitness functions did not differ significantly between morphs. Thus, although we found evidence for the intersection of morph fitness functions with respect to demography, we did not find such an intersection in relation to male body size. Our results highlight how demography can exert selection pressures shaping the evolution of the conditional strategy in species with ARTs.

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.

Environmental quality alters female costs and benefits of evolving under enforced monogamy.

BACKGROUND: Currently many habitats suffer from quality loss due to environmental change. As a consequence, evolutionary trajectories might shift due to environmental effects and potentially increase extinction risk of resident populations. Nevertheless, environmental variation has rarely been incorporated in studies of sexual selection and sexual conflict, although local environments and individuals' condition undoubtedly influence costs and benefits. Here, we utilise polyandrous and monogamous selection lines of flour beetles, which evolved in presence or absence of sexual selection for 39 generations. We specifically investigated effects of low vs. standard food quality (i.e. stressful vs. benign environments) on reproductive success of cross pairs between beetles from the contrasting female and male selection histories to assess gender effects driving fitness. RESULTS: We found a clear interaction of food quality, male selection history and female selection history. Monogamous females generally performed more poorly than polyandrous counterparts, but reproductive success was shaped by selection history of their mates and environmental quality. When monogamous females were paired with polyandrous males in the standard benign environment, females seemed to incur costs, possibly due to sexual conflict. In contrast, in the novel stressful environment, monogamous females profited from mating with polyandrous males, indicating benefits of sexual selection outweigh costs. CONCLUSIONS: Our findings suggest that costs and benefits of sexually selected adaptations in both sexes can be profoundly altered by environmental quality. With regard to understanding possible impacts of environmental change, our results further show that the ecology of mating systems and associated selection pressures should be considered in greater detail.

Annotated zoogeography of non-marine Tardigrada. Part I: Central America.

Dividing the world into nine regions, this first paper describes literature records of the limno-terrestrial tardigrades (Tardigrada) reported from Central America. Updating previously published species lists we have revised the taxonomy and provided additional habitat, geographic co-ordinates, and biogeographic comments. It is hoped this work will serve as a reference point and background for further zoogeographic studies.

Tardigrades from Peru (South America), with descriptions of three new species of Parachela.

In four samples of mosses and mosses mixed with lichens collected in the Peruvian region of Cusco, 344 tardigrades, 78 free-laid eggs and six simplexes were found. In total, nine species were identified: Cornechiniscus lobatus, Echiniscus dariae, E. ollantaytamboensis, Isohypsibius condorcanquii sp. nov., Macrobiotus pisacensis sp. nov., Milnesium krzysztofi, Minibiotus intermedius, Paramacrobiotus intii sp. nov. and Pseudechiniscus ramazzottii ramazzottii. Isohypsibius condorcanquii sp. nov. is most similar to I. baldii, but differs mainly by the absence of ventral sculpture, the presence of the oral cavity armature, a different macroplacoid length sequence and a different shape of macroplacoids. The new species also differs from other congeners by a different dorsal sculpture, the absence of cuticular bars under the claws and the absence of eyes. Macrobiotus pisacensis sp. nov. differs from the most similar M. ariekammensis and M. kirghizicus by a different oral cavity armature, the presence of cuticular pores, details of egg morphology and some morphometric characters of both animals and eggs. Paramacrobiotus intii sp. nov. differs from most similar species of the genus by a different type of the oral cavity armature, details of egg morphology and some morphometric characters of both animals and eggs. In addition, we briefly discuss the tardigrade fauna of Peru, and propose a simple and economic system of describing relative lengths of pharyngeal macroplacoids. The system is especially useful in interspecific comparisons and differential diagnoses.

Fixation offset decreases manual inhibition of return (IOR) in detection and discrimination tasks.

Attention can be covertly shifted to peripheral stimuli to improve their processing. However, attention is also then inhibited against returning to the previously attended location; thus, both detection and discrimination of a stimulus presented at that location decrease (the inhibition of return [IOR] effect). The after-effect of the covert orienting hypothesis postulates a close link between attention shifting, IOR, and oculomotor control. The fixation offset, which improves the generation of saccades, decreases IOR in detection tasks, suggesting a close link between IOR and oculomotor control. However, according to some alternative views (e.g., the input-based IOR hypothesis and the object files segregation/integration hypothesis), IOR may be related to some sensory rather than motor processes. Some studies support that view and show that IOR may occur differently in detection and discrimination tasks and that oculomotor processes do not affect IOR in tasks where manual responses are required and eye movements are suppressed. Two experiments presented in this article show that removing the fixation point decreases manual IOR in detection and discrimination tasks. The results are discussed in terms of various theoretical approaches.

Temperament Affected Visuospatial Orienting on Discrimination Tasks.

In the Posner cueing paradigm, the early attentional capture and subsequent inhibition of return (IOR) of attention to the same location, although they are microscale phenomena measured in milliseconds, seem to encapsulate the interaction between two fundamental dimensions of behavior - engaging in and sustaining activity versus withdrawing from and inhibiting activity. In the field of differential psychology, the dynamics of reciprocal relations between these behavioral dimensions have been thought to be determined by central nervous system properties that constitute an individual's temperament. Yet the research on any differential effects of temperament on visuospatial orienting is rather sparse and has produced ambiguous results. Here, we used saccadic responses to measure whether individual differences in reactivity as a temperamental trait might affect orienting of visuospatial attention on discrimination cueing tasks. Our results suggested that, in individuals with lower reactivity, attentional capture took place at a short stimulus onset asynchrony (SOA), producing a facilitatory cueing effect, which was not the case in those who were higher in reactivity. We explain and discuss these results with the Regulative Theory of Temperament.

Analysis of Nephrolithiasis Treatment in Highest Reference Hospital-Occurrence of Acromegaly in the Study Group.

BACKGROUND: Urolithiasis is one of the most common diseases of the urinary system, the incidence of which is assumed to be up to 100,000 cases per million (10% of the population). The cause of it is dysregulation of renal urine excretion. Acromegaly is a very rare endocrine disorder that causes a somatotropic pituitary adenoma producing higher amounts of growth hormone. It occurs approximately in 80 cases per million (about 0.008% of the population). One of the acromegaly complications may be urolithiasis. METHODS: Clinical and laboratory results of 2289 patients hospitalized for nephrolithiasis in the highest reference hospital were retrospectively analyzed, distinguishing a subgroup of patients with acromegaly. Statistical analysis was performed to compare the prevalence of the disease in the analyzed subgroup with the epidemiological results available in up-to-date literature. RESULTS: The distribution of nephrolithiasis treatment was definitely in favor of non-invasive and minimally invasive treatment. The methods used were as follows: ESWL (61.82%), USRL (30.62%), RIRS (4.15%), PCNL (3.1%), and pyelolithotomy (0.31%). Such a distribution limited the potential complications of the procedures while maintaining the high effectiveness of the treatment. Among two thousand two hundred and eighty-nine patients with urolithiasis, two were diagnosed with acromegaly before the nephrological and urological treatment, and seven were diagnosed de novo. Patients with acromegaly required a higher percentage of open surgeries (including nephrectomy) and also had a higher rate of kidney stones recurrence. The concentration of IGF-1 in patients with newly diagnosed acromegaly was similar to those treated with somatostatin analogs (SSA) due to incomplete transsphenoidal pituitary surgery. CONCLUSIONS: In the population of patients with urolithiasis requiring hospitalization and interventional treatment compared to the general population, the prevalence of acromegaly was almost 50-fold higher (p = 0.025). Acromegaly itself increases the risk of urolithiasis.

Green armoured tardigrades (Echiniscidae: Viridiscus), including a new species from the Southern Nearctic, exemplify problems with tardigrade variability research.

Ranges of tardigrade intraspecific and interspecific variability are not precisely defined, both in terms of morphology and genetics, rendering descriptions of new taxa a cumbersome task. This contribution enhances the morphological and molecular dataset available for the heterotardigrade genus Viridiscus by supplying new information on Southern Nearctic populations of V. perviridis, V. viridianus, and a new species from Tennessee. We demonstrate that, putting aside already well-documented cases of significant variability in chaetotaxy, the dorsal plate sculpturing and other useful diagnostic characters, such as morphology of clavae and pedal platelets, may also be more phenotypically plastic characters at the species level than previously assumed. As a result of our integrative analyses, V. viridianus is redescribed, V. celatus sp. nov. described, and V. clavispinosus designated as nomen inquirendum, and its junior synonymy with regard to V. viridianus suggested. Morphs of three Viridiscus species (V. perviridis, V. viridianus, and V. viridissimus) are depicted, and the implications for general echiniscid taxonomy are drawn. We emphasise that taxonomic conclusions reached solely through morphological or molecular analyses lead to a distorted view on tardigrade α-diversity.

Rough backs: taxonomic value of epicuticular sculpturing in the genus Milnesium Doyère, 1840 (Tardigrada: Apochela).

The phylum Tardigrada comprises ~ 1400 described species that inhabit a wide range of ecosystems throughout the globe. Tardigrades are generally considered taxonomically challenging due to a strongly limited number of taxonomically informative morphological traits and microscopic size. Of all tardigrade groups, the taxonomy of Milnesium Doyère, 1840 is particularly difficult because in comparison with most other eutardigrades, the genus lacks the taxonomically informative egg shell ornamentation and/or placoids in the muscle pharynx. Therefore, any new morphological traits that could be used in species delineation and identification are priceless. In this contribution, we review and evaluate taxonomic value of the dorsal cuticle morphology. Specifically, by means of experimental taxonomy, we demonstrate the first extreme case of ontogenetic variability in dorsal epicuticle sculpturing using a new species from Portugal, Milnesium decorum sp. nov. Furthermore, we verify the existence of dorsal gibbosities in Milnesium reticulatum Pilato, Binda, Lisi, 2002, the only species of the genus allegedly exhibiting these structures. Finally, we provide a diagnostic key to the Milnesium granulatum morphogroup.