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.

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.

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.

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.

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.

First insights into female sperm storage duration in tardigrades.

Female sperm storage is ubiquitous in the animal kingdom and it has been shown to be linked to several evolutionary processes, from postcopulatory sexual selection to dispersal. Here we report, for the first time, long-term sperm storage in females of the tardigrade Macrobiotus polonicus. Females, isolated after a short contact with a male, were able to use the stored sperm for up to 5 weeks (mean of 2 weeks), which translates to a considerable proportion of female post-mating longevity under controlled laboratory conditions (60% on average). Our study provides the first insights into the duration of sperm storage, an underexplored feature of the reproductive biology of tardigrades. Additionally, we discuss important considerations for reproductive studies on these non-model animals.

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.

The importance of being integrative: a remarkable case of synonymy in the genus Viridiscus (Heterotardigrada: Echiniscidae).

There are two predominant sources of taxonomically useful morphological variability in the diverse tardigrade family Echiniscidae: the internal structure and surface sculpture of the cuticular plates covering the dorsum (sculpturing) and the arrangement and morphology of the trunk appendages (chaetotaxy). However, since the appendages often exhibit intraspecific variation (they can be reduced or can develop asymmetrically), sculpturing has been considered more stable at the species level and descriptions of new echiniscid species based solely on morphology are still being published. Here, we present a case study in which a detailed analysis of the morphology and multiple genetic markers of several species of the genus Viridiscus shows that cuticular sculpture may also exhibit considerable intraspecific variation and lead to false taxonomic conclusions. In a population collected from the eastern Nearctic, in the type locality of the recently described species V. miraviridis, individuals with transitional morphotypes between those reported for V. viridissimus and V. miraviridis were found. Importantly, all morphotypes within the viridissimus-miraviridis spectrum were grouped in a single monospecific clade according to rapidly evolving markers (ITS-1, ITS-2 and COI). Given the morphological and genetic evidence, we establish V. miraviridis as a junior synonym of V. viridissimus. This study explicitly demonstrates that a lack of DNA data associated with morphological descriptions of new taxa jeopardizes the efforts to unclutter tardigrade systematics. Additionally, V. perviridis and V. viridissimus are reported from Lâm Dong Province in southern Vietnam, which considerably broadens their known geographic ranges.