Recovery from anhydrobiosis in the tardigrade Paramacrobiotus experimentalis: Better to be young than old and in a group than alone.

Desiccation-tolerant organisms can survive dehydration in a state of anhydrobiosis. Tardigrades can recover from anhydrobiosis at any life stage and are considered among the toughest animals on Earth. However, the factors that influence recovery from anhydrobiosis are not well understood. The study aimed to evaluate the effect of sex, age, the presence of other individuals and the combination of the number and duration of anhydrobiosis episodes on the recovery of Paramacrobiotus experimentalis. The activity of 1200 individuals for up to 48 h after rehydration was evaluated using analysis of variance (ANOVA). Age was the main factor influencing return to activity, followed by the combination of number and duration of anhydrobiosis episodes, influence of the presence of other individuals, and sex. More individuals returned to activity after repeated short than repeated long anhydrobiosis episodes and older individuals were less likely to recover than younger individuals. In addition, when compared to single animals, the presence of other individuals resulted in higher number of active animals after dehydration and rehydration. The effect of sex was significant, but there was no general tendency for one sex to recover from anhydrobiosis better than the other one. The results contribute to a better understanding of the anhydrobiosis ability of Paramacrobiotus experimentalis and provide background for full explanation of molecular, cellular and environmental mechanisms of anhydrobiosis.

Potential oxidative stress related targets of mitochondria-focused therapy of PTSD.

Post-traumatic stress disorder (PTSD) remains a highly prevalent, under-diagnosed, and under-treated psychiatric disorder that often deteriorates over time, and is highly comorbid with major depressive disorder, suicidality, and substance use disorder. Several biomarkers have been proposed but have yet to be implemented into clinical practice. Treatments, including selective serotonin reuptake inhibitors, are efficacious in only a small number of patients, which underscores the need to develop novel, efficient treatments. Mitochondrial dysfunction resulting from chronic oxidative stress has been linked with both altered neurotransmitter signaling and the inflammatory response. Hereinafter, we discuss mechanisms by which mitochondrial dysfunction may contribute to the development of PTSD symptoms, and how these may even increase PTSD susceptibility. We also highlight possible therapeutic targets to reduce oxidative stress to prevent or treat PTSD symptoms.

The effect of hypomagnetic field on survival and mitochondrial functionality of active Paramacrobiotus experimentalis females and males of different age.

Even for tardigrades, often called the toughest animals on Earth, a hypomagnetic field (HMF) is an extreme environment. However, studies on the effect of HMF on tardigrades and other invertebrates are scarce. Mitochondria play an important role in an organism's response to extreme conditions. The effect of HMF on the mitochondrial inner membrane potential (Δψ), a well-known marker of mitochondria functionality, shows that mitochondria are very sensitive to HMF. To measure the HMF effect on Paramacrobiotus experimentalis, we calculated the tardigrade survival rate and Δψ level after HMF treatments of different durations. We also estimated the relationship between the age and sex of the tardigrade and the HMF effect. We observed age- and sex-related differences in Δψ and found that Δψ changes after HMF treatment were dependent on its duration as well as the animal's age and sex. Furthermore, active P. experimentalis individuals displayed a high survival rate after HMF treatment. The data may contribute to the understanding of tardigrade aging and their resistance to extreme conditions including HMF, which in turn may be useful for future space explorations.

VDAC as a Cellular Hub: Docking Molecules and Interactions.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane [...].

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

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

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

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

Redox-Sensitive VDAC: A Possible Function as an Environmental Stress Sensor Revealed by Bioinformatic Analysis.

Voltage-dependent anion-selective channel (VDAC) allows the exchange of small metabolites and inorganic ions across the mitochondrial outer membrane. It is involved in complex interactions that regulate mitochondrial and cellular functioning. Many organisms have several VDAC paralogs that play distinct but poorly understood roles in the life and death of cells. It is assumed that such a large diversity of VDAC-encoding genes might cause physiological plasticity to cope with abiotic and biotic stresses known to impact mitochondrial function. Moreover, cysteine residues in mammalian VDAC paralogs may contribute to the reduction-oxidation (redox) sensor function based on disulfide bond formation and elimination, resulting in redox-sensitive VDAC (rsVDAC). Therefore, we analyzed whether rsVDAC is possible when only one VDAC variant is present in mitochondria and whether all VDAC paralogs present in mitochondria could be rsVDAC, using representatives of currently available VDAC amino acid sequences. The obtained results indicate that rsVDAC can occur when only one VDAC variant is present in mitochondria; however, the possibility of all VDAC paralogs in mitochondria being rsVDAC is very low. Moreover, the presence of rsVDAC may correlate with habitat conditions as rsVDAC appears to be prevalent in parasites. Thus, the channel may mediate detection and adaptation to environmental conditions.

How the Geomagnetic Field Influences Life on Earth - An Integrated Approach to Geomagnetobiology.

Earth is one of the inner planets of the Solar System, but - unlike the others - it has an oxidising atmosphere, relatively stable temperature, and a constant geomagnetic field (GMF). The GMF does not only protect life on Earth against the solar wind and cosmic rays, but it also shields the atmosphere itself, thus creating relatively stable environmental conditions. What is more, the GMF could have influenced the origins of life: organisms from archaea to plants and animals may have been using the GMF as a source of spatial information since the very beginning. Although the GMF is constant, it does undergo various changes, some of which, e.g. a reversal of the poles, weaken the field significantly or even lead to its short-term disappearance. This may result in considerable climatic changes and an increased frequency of mutations caused by the solar wind and cosmic radiation. This review analyses data on the influence of the GMF on different aspects of life and it also presents current knowledge in the area. In conclusion, the GMF has a positive impact on living organisms, whereas a diminishing or disappearing GMF negatively affects living organisms. The influence of the GMF may also be an important factor determining both survival of terrestrial organisms outside Earth and the emergence of life on other planets.

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

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

The Diversity of the Mitochondrial Outer Membrane Protein Import Channels: Emerging Targets for Modulation.

The functioning of mitochondria and their biogenesis are largely based on the proper function of the mitochondrial outer membrane channels, which selectively recognise and import proteins but also transport a wide range of other molecules, including metabolites, inorganic ions and nucleic acids. To date, nine channels have been identified in the mitochondrial outer membrane of which at least half represent the mitochondrial protein import apparatus. When compared to the mitochondrial inner membrane, the presented channels are mostly constitutively open and consequently may participate in transport of different molecules and contribute to relevant changes in the outer membrane permeability based on the channel conductance. In this review, we focus on the channel structure, properties and transported molecules as well as aspects important to their modulation. This information could be used for future studies of the cellular processes mediated by these channels, mitochondrial functioning and therapies for mitochondria-linked diseases.

Mitochondrial alternative oxidase contributes to successful tardigrade anhydrobiosis.

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

The Open State Selectivity of the Bean Seed VDAC Depends on Stigmasterol and Ion Concentration.

The voltage-dependent anion channel (VDAC) is the major pathway for metabolites and ions transport through the mitochondrial outer membrane. It can regulate the flow of solutes by switching to a low conductance state correlated with a selectivity reversal, or by a selectivity inversion of its open state. The later one was observed in non-plant VDACs and is poorly characterized. We aim at investigating the selectivity inversion of the open state using plant VDAC purified from Phaseolus coccineus (PcVDAC) to evaluate its physiological role. Our main findings are: (1) The VDAC selectivity inversion of the open state occurs in PcVDAC, (2) Ion concentration and stigmasterol affect the occurrence of the open state selectivity inversion and stigmasterol appears to interact directly with PcVDAC. Interestingly, electrophysiological data concerning the selectivity inversion of the PcVDAC open state suggests that the phenomenon probably does not have a significant physiological effect in vivo.

Mitochondrial Processes during Early Development of Dictyostelium discoideum: From Bioenergetic to Proteomic Studies.

The slime mold Dictyostelium discoideum's life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria's structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

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

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

Integrative description of five Pseudechiniscus species (Heterotardigrada: Echiniscidae: the suillus-facettalis complex).

Pseudechiniscus is a morphologically homogeneous heterotardigrade genus with a relatively low number of morphological features useful for the species discrimination. The species of the Pseudechiniscus suillus-facettalis complex are some of the most challenging tardigrades to identify. Here, we examine several populations from Antarctica, Italy, Madagascar and Norway that would have most likely been attributed to Pse. suillus prior to the recent redescription of the species. Populations were analysed using integrative taxonomy-a combination of classical morphology and morphometry, as well as genetic data. Besides minute differences in dorsal sculpture and morphometry, we found clear, species-specific differences in ventral sculpture which are very useful in discrimination of Pseudechiniscus species. Based on morphology (mainly ventral sculpture) and significant genetic distances in COI and ITS-2 sequences, we describe five new species: Pse. angelusalas sp. nov. from Madagascar, Pse. dastychi sp. nov. from Antarctica, Pse. ehrenbergi sp. nov. from Italy as well as Pse. indistinctus sp. nov. and Pse. lacyformis sp. nov. from Norway. Finally, we provide an updated phylogenetic tree of the genus Pseudechiniscus based on COI sequences.

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

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

Two new species of the genus Milnesium Doyère, 1840 (Tardigrada, Apochela, Milnesiidae) from Madagascar.

The knowledge of the diversity and distribution of tardigrades on Madagascar is rather poor. To date, only 13 tardigrade taxa have been reported from this region (including one Milnesium species). We examined 46 specimens belonging to two new-to-science species of the genus Milnesium described herein using an integrative approach, including classical morphology and molecular marker (COI, ITS-2 and 28S rRNA) analysis. The species were found in two moss and lichen samples collected in the Ivohibory forest in Fianarantsoa Province. Milnesium matheusi sp. nov., with claw configuration [3-3]-[3-3] and rather wide buccal tube, morphologically is most similar to: Mil. beatae Roszkowska, Ostrowska & Kaczmarek, 2015, Mil. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014, Mil. eurystomum Maucci, 1991, Mil. shilohae Meyer, 2015 and Mil. tumanovi Pilato, Sabella & Lisi, 2016; however, it differs from these by morphometric characteristics. Milnesium wrightae sp. nov., by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919. However, Mil. wrightae sp. nov. differs from Mil. quadrifidum by claw configuration ([4-4]-[4-4] in Mil. quadrifidum vs. [3-3]-[4-4] in Mil. wrightae sp. nov.), but also by the position of the fourth points on secondary branches of claws IV, which are located near the base of the claw in the new species and near the top of the claw in Mil. quadrifidum. Genotypic analysis showed that Mil. matheusi sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. t. tardigradum (ITS-2) while Mil. wrightae sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. matheusi (ITS-2). Five Milnesium taxa are recorded from the African region, including the two new species from Madagascar reported in this study.

The The TOB/SAM complex composition in mitochondria of Dictyostelium discoideum during progression from unicellularity to multicellularity.

Despite its complex life cycle including unicellular and multicellular stages, the slime mold Dictyostelium discoideum, a well-known model in biomedical research, has not been used as a model organism in studies on mitochondrial import, including its significance in cellular processes. Moreover, data concerning mitochondrial protein import machinery in D. discoideum mitochondria is limited and nothing is known about the impact of that machinery on slime mold life cycle. Here, we focused on the TOB/SAM (topogenesis of the mitochondrial outer membrane ß-barrel proteins/sorting and assembly machinery) complex. This complex is localized in the mitochondrial outer membrane and is indispensable for the formation of metabolite exchange and protein import pathways in the membrane, and substantially contributes to the regulation of mitochondrial morphology and distribution. Furthermore, the available data suggests that the TOB/SAM complex variants differ between mitochondria of multicellular and unicellular eukaryotes. Therefore, we decided to determine these variants of the TOB/SAM in mitochondria of D. discoideum progressing from single cells to early multicellular stages, when the cells stream together to form a multicellular organism. The results revealed two complex variants of the TOB/SAM complex of about 160 and 600 kDa molecular weight, present in mitochondria of D. discoideum cells at the studied stages. The discussed complex variants resemble the ones that have been already detected for the yeast Saccharomyces cerevisiae, fungus Neurospora crassa and human cells, and one of investigated variants differentiates unicellular and initial multicellular stages of the D. discoideum life cycle.

Insight into the Phytoremediation Capability of Brassica juncea (v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity.

Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Brassica species are known to display hyperaccumulation capability. Brassica juncea (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 µM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all B. juncea plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of B. juncea in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.