Analysis of the trehalose synthesis pathway of Physarum polycehalum.

Desiccation is a severe survival problem for organisms. We have been studying the desiccation tolerance mechanisms in the true slime mold Physarum polycephalum. We measured the trehalose content of P. polycephalum vegetative cells (plasmodia) and drought cells (sclerotia). Surprisingly, we found that the content in sclerotia was about 473-fold greater than in the plasmodia. We then examined trehalose metabolism-related genes via RNAseq, and consequently found that trehalose 6-phosphate phosphorylase (T6pp) expression levels increased following desiccation. Next, we cloned and expressed the genes for T6pp, trehalose 6-phosphate synthase/phosphatase (Tps/Tpp), maltooligosyltrehalose trehalohydrolase (TreZ), and maltooligosyltrehalose synthase (TreY) in E. coli. Incidentally, TreY and TreZ clones have been reported in several prokaryotes, but not in eukaryotes. This report in P. polycephalum is the first evidence of their presence in a eukaryote species. Recombinant T6pp, TreY, and TreZ were purified and confirmed to be active. Our results showed that these enzymes catalyze reactions related to trehalose production, and their reaction kinetics follow the Michaelis-Menten equation. The t6pp mRNA levels of the sclerotia were about 15-fold higher than in the plasmodia. In contrast, the expression levels of TreZ and TreY showed no significant change between the sclerotia and plasmodia. Thus, T6pp is probably related to desiccation tolerance, whereas the contribution of TreY and TreZ is insufficient to account for the considerable accumulation of trehalose in sclerotia.

Life cycle and plasmodial axenic culture of Physarum galbeum.

Myxogastrea is a group of eukaryotic microorganisms included in Amoebozoa. Its life cycle includes two trophic stages: plasmodia and myxamoeflagellates. However, only about 102 species have their complete life cycle known in literature and only about 18 species have their plasmodial axenic culture accomplished in laboratory conditions. The research presented herein involved culturing of Physarum galbeum on the water agar medium. The events that transpired during its life cycle including spore germination, plasmodia formation, and sporocarp development were documented especially the subglobose or discoid sporotheca and the stalk formation. The spores germinated by the V-shape split method to release a single protoplasm. Yellow-green pigmented phaneroplasmodia developed into sporocarps by subhypothallic type. The present article gives details of the sporocarp development of P. galbeum and its plasmodial axenic culture on solid and liquid mediums.

Diversity of bacterial communities in the plasmodia of myxomycetes.

BACKGROUND: Myxomycetes are a group of eukaryotes belonging to Amoebozoa, which are characterized by a distinctive life cycle, including the plasmodium stage and fruit body stage. Plasmodia are all found to be associated with bacteria. However, the information about bacteria diversity and composition in different plasmodia was limited. Therefore, this study aimed to investigate the bacterial diversity of plasmodia from different myxomycetes species and reveal the potential function of plasmodia-associated bacterial communities. RESULTS: The bacterial communities associated with the plasmodia of six myxomycetes (Didymium iridis, Didymium squamulosum, Diderma hemisphaericum, Lepidoderma tigrinum, Fuligo leviderma, and Physarum melleum) were identified by 16S rRNA amplicon sequencing. The six plasmodia harbored 38 to 52 bacterial operational taxonomic units (OTUs) that belonged to 7 phyla, 16 classes, 23 orders, 40 families, and 53 genera. The dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria. Most OTUs were shared among the six myxomycetes, while unique bacteria in each species only accounted for a tiny proportion of the total OTUs. CONCLUSIONS: Although each of the six myxomycetes plasmodia had different bacterial community compositions, a high similarity was observed in the plasmodia-associated bacterial communities' functional composition. The high enrichment for gram-negative (> 90%) and aerobic (> 99%) bacteria in plasmodia suggest that myxomycetes may positively recruit certain kinds of bacteria from the surrounding environment.

Electrophysiological evidence for inhibition hypothesis of micro-expressions based on tensor component analysis and Physarum network algorithm.

The inhibition hypothesis advocated by Ekman (1985) states when an emotion is concealed or masked, the true emotion is manifested as a micro-expression (ME) which is a fleeting expression lasting for 40 to 500 ms. However, research about the inhibition hypothesis of ME from the perspective of electrophysiology is lacking. Here, we report the electrophysiological evidence obtained from an electroencephalography (EEG) data analysis method. Specifically, we designed an ME elicitation paradigm to collect data of MEs of positive emotions and EEG from 70 subjects, and proposed a method based on tensor component analysis (TCA) combined with the Physarum network (PN) algorithm to characterize the spatial, temporal, and spectral signatures of dynamic EEG data of MEs. The proposed TCA-PN methods revealed two pathways involving dorsal and ventral streams in functional brain networks of MEs, which reflected the inhibition processing and emotion arousal of MEs. The results provide evidence for the inhibition hypothesis from an electrophysiological standpoint, which allows us to better understand the neural mechanism of MEs.

High Environmentally Induced Plasticity in Spore Size and Numbers of Nuclei per Spore in Physarum albescens (Myxomycetes).

Spore size enables dispersal in plasmodial slime molds (Myxomycetes) and is an important taxonomic character. We recorded size and the number of nuclei per spore for 39 specimens (colonies of 50-1000 sporocarps) of the nivicolous myxomycete Physarum albescens, a morphologically defined taxon with several biological species. For each colony, three sporocarps were analyzed from the same spore mount under brightfield and DAPI-fluorescence, recording ca. 14,000 spores per item. Diagrams for spore size distribution showed narrow peaks of mostly uninucleate spores. Size was highly variable within morphospecies (10.6-13.5 µm, 11-13%), biospecies (3-13%), even within spatially separated colonies of one clone (ca. 8%); but fairly constant for a colony (mean variation 0.4 µm, ca. 1.5%). ANOVA explains most of this variation by the factor locality (within all colonies: 32.7%; within a region: 21.4%), less by biospecies (13.5%), whereas the contribution of intra-colony variation was negligible (<0.1%). Two rare aberrations occur: 1) multinucleate spores and 2) oversized spores with a double or triple volume of normal spores. Both are not related to each other or limited to certain biospecies. Spore size shows high phenotypic plasticity, but the low variation within a colony points to a strong genetic background.

Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing.

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1-3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes.

Monte Carlo Physarum Machine: Characteristics of Pattern Formation in Continuous Stochastic Transport Networks.

We present Monte Carlo Physarum Machine (MCPM): a computational model suitable for reconstructing continuous transport networks from sparse 2D and 3D data. MCPM is a probabilistic generalization of Jones's (2010) agent-based model for simulating the growth of Physarum polycephalum (slime mold). We compare MCPM to Jones's work on theoretical grounds, and describe a task-specific variant designed for reconstructing the large-scale distribution of gas and dark matter in the Universe known as the cosmic web. To analyze the new model, we first explore MCPM's self-patterning behavior, showing a wide range of continuous network-like morphologies-called polyphorms-that the model produces from geometrically intuitive parameters. Applying MCPM to both simulated and observational cosmological data sets, we then evaluate its ability to produce consistent 3D density maps of the cosmic web. Finally, we examine other possible tasks where MCPM could be useful, along with several examples of fitting to domain-specific data as proofs of concept.

Phylogeny of Physarida (Amoebozoa, Myxogastria) Based on the Small-Subunit Ribosomal RNA Gene, Redefinition of Physarum pusillum s. str. and Reinstatement of P. gravidum Morgan.

Myxomycetes (also called Myxogastria or colloquially, slime molds) are worldwide occurring soil amoeboflagellates. Among Amoebozoa, they have the notable characteristic to form, during their life cycle, macroscopic fruiting bodies, that will ultimately release spores. Some 1,000 species have been described, based on the macroscopic and microscopic characteristics of their fruiting bodies. We were interested in Physarum pusillum (Berk. & M.A. Curtis) G. Lister, a very common species described with two variants, each bearing such morphological differences that they could represent two distinct species. In order to test this, we observed key characters in a large selection of specimens attributed to P.  pusillum, to its synonyms (in particular Physarum gravidum), and to related species. In addition, the small-subunit ribosomal RNA gene was obtained from seven of these specimens. Based on these data, we provide a comprehensive phylogeny of the order Physarida (Eukaryota: Amoebozoa: Conosa: Macromycetozoa: Fuscisporidia). Morphology and phylogeny together support the reinstatement of P. gravidum Morgan 1896 with a neotype here designated, distinct from P. pusillum, here redefined.

Profiles of Physarum Microplasmodial Phosphatase Activity Crucial to Cytoplasmic Streaming and Spherule Formation.

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.

Active poroelastic two-phase model for the motion of physarum microplasmodia.

The onset of self-organized motion is studied in a poroelastic two-phase model with free boundaries for Physarum microplasmodia (MP). In the model, an active gel phase is assumed to be interpenetrated by a passive fluid phase on small length scales. A feedback loop between calcium kinetics, mechanical deformations, and induced fluid flow gives rise to pattern formation and the establishment of an axis of polarity. Altogether, we find that the calcium kinetics that breaks the conservation of the total calcium concentration in the model and a nonlinear friction between MP and substrate are both necessary ingredients to obtain an oscillatory movement with net motion of the MP. By numerical simulations in one spatial dimension, we find two different types of oscillations with net motion as well as modes with time-periodic or irregular switching of the axis of polarity. The more frequent type of net motion is characterized by mechano-chemical waves traveling from the front towards the rear. The second type is characterized by mechano-chemical waves that appear alternating from the front and the back. While both types exhibit oscillatory forward and backward movement with net motion in each cycle, the trajectory and gel flow pattern of the second type are also similar to recent experimental measurements of peristaltic MP motion. We found moving MPs in extended regions of experimentally accessible parameters, such as length, period and substrate friction strength. Simulations of the model show that the net speed increases with the length, provided that MPs are longer than a critical length of ≈ 120 µm. Both predictions are in line with recent experimental observations.

Physarum inspires research beyond biomimetic algorithms: Reply to comments on "Does being multi-headed make you better at solving problems?"

We look at a recent expansion of Physarum research from inspiring biomimetic algorithms to serving as a model organism in the evolutionary study of perception, memory, learning, and decision making.

A Physarum Centrality Measure of the Human Brain Network.

The most important goals of brain network analyses are to (a) detect pivotal regions and connections that contribute to disproportionate communication flow, (b) integrate global information, and (c) increase the brain network efficiency. Most centrality measures assume that information propagates in networks with the shortest connection paths, but this assumption is not true for most real networks given that information in the brain propagates through all possible paths. This study presents a methodological pipeline for identifying influential nodes and edges in human brain networks based on the self-regulating biological concept adopted from the Physarum model, thereby allowing the identification of optimal paths that are independent of the stated assumption. Network hubs and bridges were investigated in structural brain networks using the Physarum model. The optimal paths and fluid flow were used to formulate the Physarum centrality measure. Most network hubs and bridges are overlapped to some extent, but those based on Physarum centrality contain local and global information in the superior frontal, anterior cingulate, middle temporal gyrus, and precuneus regions. This approach also reduced individual variation. Our results suggest that the Physarum centrality presents a trade-off between the degree and betweenness centrality measures.

Liquid brains, solid brains.

Cognitive networks have evolved a broad range of solutions to the problem of gathering, storing and responding to information. Some of these networks are describable as static sets of neurons linked in an adaptive web of connections. These are 'solid' networks, with a well-defined and physically persistent architecture. Other systems are formed by sets of agents that exchange, store and process information but without persistent connections or move relative to each other in physical space. We refer to these networks that lack stable connections and static elements as 'liquid' brains, a category that includes ant and termite colonies, immune systems and some microbiomes and slime moulds. What are the key differences between solid and liquid brains, particularly in their cognitive potential, ability to solve particular problems and environments, and information-processing strategies? To answer this question requires a new, integrative framework. This article is part of the theme issue 'Liquid brains, solid brains: How distributed cognitive architectures process information'.

The life cycles of two species of Myxomycetes in Physarales, Physarum rigidum and Didymium squamulosum.

Myxomycetes are eukaryotic microorganisms containing characteristics akin to both fungi and amoebae. They can complete their whole life cycles while being cultured on agar media, and under-laboratory conditions, which favors taxonomic, phylogenetic, and cytological researches. Here, we describe the life cycles of two such species: Didymium squamulosum collected from the field and Physarum rigidum cultured from moist chamber both belonging to the Order Physarales. Three per cent oat-agar media (OAM) was used to culture the plasmodia until they aggregated and were almost starved. Natural light was then applied to the plasmodia to induce fructification. Their life cycles share the same common stages, namely: spore, myxamoebae, swarm cell, plasmodia, and sporulation. In this study, we describe the morphogenesis from spore to spore of two species by differential interference contrast (DIC) and stereoscopic microscopies, as well as discuss the differences between the development of both species and interspecies. We found that the spore germination method of both species was the same. However, there were differences noted in time taken and fruiting body formation. Unlike P. rigidum, the species D. squamulosum did not require natural light stimulation. Moreover, the maturation process of both species had similar color transitions but exhibited distinct morphology in each developmental stage except during the swarm cell stage.

Developing a multispectral model for detection of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) changes in fish fillet using physarum network and genetic algorithm (PN-GA) method.

A multispectral model for the detection of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) changes in grass carp and salmon fillet was developed using physarum network and genetic algorithm (PN-GA) method for the first time. Partial least-squares regression (PLSR), multiple linear regressions (MLR), and principal component regression (PCR) algorithms were used to predict the DHA and EPA using optimal wavelengths selected by PN-GA. The MLR models showed the best DHA prediction results for both grass carp and salmon fillets, and also showed good prediction for EPA in grass carp fillet but poor prediction in salmon fillet. The MLR models were then applied for visualizing the spatial distribution of DHA and EPA changes in two fish fillets. The current results demonstrated that a developed multispectral imaging system could be feasibly constructed for DHA and EPA measurement in fish species with the optimal wavelengths selected by PN-GA method.

Cell learning.

Although we often think of cells as small, simple building blocks of life, in fact they are highly complex and can perform a startling variety of functions. In our bodies, cells are programmed by complex differentiation pathways and are capable of responding to a bewildering range of chemical and physical signals. Free-living single-celled organisms, such as bacteria or protists, have to cope with varying environments, locate prey and potential mates, and escape from predators - all of the same tasks that a free-living animal is faced with. When animals face complex behavioral challenges, they rely on their cognitive abilities - the ability to learn from experience, to analyse a situation and choose an appropriate course of action. This ability is essential for survival and should, in principle, be a ubiquitous feature of all living things regardless of the complexity of the organism.

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3' ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3' end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3' ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3' ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3' ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3' end. Optimization of this activity is obtained through alteration of the lengths of the 5'-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.

Morphological and molecular characterization of a new succulenticolous Physarum (Myxomycetes, Amoebozoa) with unique polygonal spores linked in chains.

A new plasmodiocarpic and sporocarpic species of myxomycete in the genus Physarum is described and illustrated. This new species appeared on decayed leaves and remains of succulent plants (Agave, Opuntia, Yucca) growing in arid zones. It differs from all other species in the genus in having polyhedral spores linked in chains like a string of beads, a unique feature within all known myxomycetes. Apart from detailed morphological data, partial sequences of both the small-subunit ribosomal RNA and elongation factor 1-alpha genes, generated from four isolates collected in two distant regions, i.e., Mexico and Canary Islands, are also provided in this study. Combined evidence supports the identity of the specimens under study as a new species.