Brainless but smart: Investigating cognitive-like behaviors in the acellular slime mold physarum polycephalum

Evolutionary pressures to improve fitness, have enabled living systems to make adaptive decisions when faced with heterogeneous and changing environmental and physiological conditions. This dissertation investigated the mechanisms of how environmental and physiological factors affect the behaviors of non-neuronal organisms. The acellular slime mold Physarum polycephalum was used as the model organism, which is a macroscopic, unicellular organism, that self-organizes into a network of intersecting tubules. Without using neurons, P. polycephalum can solve labyrinth mazes, build efficient tubule networks, and make adaptive decisions when faced with complicated trade-offs, such as between food quality and risk, speed and accuracy, and exploration and exploitation. However, the understanding of the mechanisms used by P. polycephalum in exhibiting such behaviors is very limited. Therefore, the objective of this dissertation is to understand the mechanisms adopted by non-neuronal organisms to explore and exploit resources in the physical environment, using environmental and physiological information. To this end, the dissertation characterizes the direction and amount of influence between different regions of tubule-shaped P. polycephalum cells in binary food choice experiments. The results show that when the two food sources are identical in quality, the regions near the food source act as the drivers of P. polycephalum tubule behavior. Conversely, when one of the food sources is more enriched with nutrients, the regions near the rejected food source were found to drive the tubule behavior. Secondly, a generalized choice-making criterion was formulated to determine the choice-making behaviors of P. polycephalum, examine whether sufficient experimental time was given to make a choice, and determine the time point at which a choice was made. The criterion was tested on binary food choice experiments using P. polycephalum tubules. The results show that P. polycephalum made a choice for the option for the better food option, except when the differences in food quality were low. Moreover, the criterion was found to not determine the choice-making behaviors when the food sources presented were identical in quality. Thirdly, the dissertation investigated whether P. polycephalum cells modify their future exploratory behavior using their past foraging experience. The results did not find a strong influence of the past foraging experience on the exploratory networks formed by P. polycephalum cells. Finally, P. polycephalum exploratory behaviors were examined and compared when the cells were in high-energy versus low-energy physiological conditions. Interestingly, the study found the P. polycephalum cells in low-energy conditions show an increased tendency to split themselves into multiple autonomous cells. Additionally, the behavior is shown to increase the fitness of the cell by increasing its foraging efficiency

Sporulation competence in Physarum polycephalum CL

The central aim of this work was to explore the possible value of P. polycephalum CL as a model of eukaryotic differentiation. Initially it was hoped to obtain a method of reproducibly obtaining sporulation and later to analyse sporulation both biochemically and genetically. Microplasmodia of the CL strain were found to yield the highest sporulation frequencies when harvested at the end of exponential growth. Sporulation frequencies of microplasmodia harvested at other points in the growth cycle could be improved by allowing overnight growth, as a surface plasmodium, before exposure to starvation medium. The minimum length of starvation in the dark required before one light pulse would induce sporulation of all plasmodia was found to be 72 h. Plasmodia became committed to sporulation about 4 h after illumination. The time of commitment to sporulation was related to the time of illumination, and not the overall length of starvation, since altering the time of illumination also altered the time of commitment. The importance of DNA replication and mitosis during the process of sporulation was assessed by examining the effects of inhibitors of these events on starving plasmodia. Nocodazole, an inhibitor of microtubule assembly, prevented sporulation if added any time up to 48 h during starvation. It was assumed, therefore, that the last mitosis during starvation occurred at about 48 h. However, nocodazole might also have affected some other event, besides mitosis, which involved microtubules. The DNA synthesis inhibitor, hydroxyurea, prevented sporulation if added at any time up to 24 h during starvation. This suggested that the last DNA replication preceded the last event susceptible to inhibition by nocodazole by some 24 h. By pulse labelling with methyl-HJ thymidine during starvation, the periods of DNA synthesis during the 72 h starvation period were defined. Periods of DNA replication began at about 4 h, 15 h and 24 h during starvation, confirming that the last replication occurred at about 24 h and demonstrating that this was the third replication to occur during starvation. The patterns of DNA replication in a sporogenous and an asporogenous culture were compared in an attempt to further clarify the role of DNA replication during sporulation. In the asporogenous derivative, the third period of DNA synthesis, normally detected in the wild type strain, did not occur, yet the previous two rounds of DNA synthesis took place normally. The asporogenous strain was produced by continuous subculture of microplasmodia in broth medium. Before the strain became fully asporogenous, it showed a delay in response to light before it would sporulate. Thus the strain only sporulated after a light pulse at 96 h instead of the normal 72 h. There was, in this strain, a concomitant delay in the escape of the plasmodium from nocodazole inhibition of sporulation. Thus the final replication at about 24 h during starvation and the nocodazole sensitive event which followed some 24 h later were important for determining the condition of the plasmodium for response to light and advance to sporulation. Microplasmodial cultures were able to grow in the presence of both hydroxyurea and the protein synthesis inhibitor, cycloheximide, at concentrations which normally inhibited growth, if incubated in their presence for extended lengths of time. This may have been due to instability of the drugs at the incubation temperature or P. polyc6phalum may have been capable of breaking these drugs down. Similar results were obtained with nocodazole, but in addition micro-plasmodia also developed resistance to the drug. A method of isolating sporulation deficient mutants was developed and several such mutants were obtained. In a preliminary genetic analysis of sporulation a cross between sporogenous (Spo+) amoebae and asporogenous (Spo-) amoebae was made. Although the diploid Spo+/Spo- plasmodium sporulated, none of the progeny able to form plasmodia in clones (matAh) were able to do so. Sporulation capacity of Spo+/Spo- heterokaryons formed by fusion of plasmodia was also investigated, in these asporogeny was dominant. Although no definite results were obtained from the genetic aspect of this work it has provided a base for further genetical studies on the process of sporulation in P. polycephalum CL

Representation of shape mediated by environmental stimuli in Physarum polycephalum and a multi-agent model

© 2015 Taylor & Francis. The slime mould Physarum polycephalum is known to construct protoplasmic transport networks which approximate proximity graphs by foraging for nutrients during its plasmodial life cycle stage. In these networks, nodes are represented by nutrients and edges are represented by protoplasmic tubes. These networks have been shown to be efficient in terms of length and resilience of the overall network to random damage. However, relatively little research has been performed in the potential for Physarum transport networks to approximate the overall shape of a data-set. In this paper we distinguish between connectivity and shape of a planar point data-set and demonstrate, using scoping experiments with plasmodia of P. polycephalum and a multi-agent model of the organism, how we can generate representations of the external and internal shapes of a set of points. As with proximity graphs formed by P. polycephalum, the behaviour of the plasmodium (real and model) is mediated by environmental stimuli. We further explore potential morphological computation approaches with the multi-agent model, presenting methods which approximate the Convex Hull and the Concave Hull. We demonstrate how a growth parameter in the model can be used to transition between Convex and Concave Hulls. These results suggest novel mechanisms of morphological computation mediated by environmental stimuli

The transition between growth and sporulation in Physarum polycephalum CL

The overall aim of this project was to examine the relationship between the cell cycle and sporulation in Physarum polycephalum: in particular, to determine when sporulation specific genes were transcribed. The work fell into three sections. The first made use of the anti-fungal agent nocodazole to determine if a plasmodium competent to sporulate after 72 h starvation was in the G1 or the G2 phase of the cell cycle. Clear evidence was obtained that differentiation was initiated from the G2 phase of the cell cycle. The effect of blocking mitosis with nocodazole on the subsequent DNA replication was investigated in both growing and starving plasmodia. It had previously been reported, and was shown in this work, that P. polycephalum has no G1 phase in the cell cycle during growth. The fact that growing and starving plasmodia responded similarly to nocodazole with regard to the onset of DNA synthesis indicated that there was no prolonged G1 period during the starvation period in P. polycephalum. It is postulated that nocodazole may interfere with a temperature-sensitive pathway that controls both the increase in thymidine kinase activity and metaphase onset. The second part of the investigation was to approach the problem of pinpointing when in the G2 phase of the cell cycle, there was sporulation specific transcription. It was assumed that this question might be answered by differential screening of a genomic library of P. polycephalum, using as probes radiolabelled copy DNA prepared from poly(A)+ RNA from growing and starving plasmodia. The first requirement was a genomic library of P. polycephalum CL DNA. Of the two phage vectors, Charon 4AP and lambda1059 which were compared, the latter proved to be superior as it was shown that a genomic library prepared in Charon 4AP would be diluted by the presence of a considerable number of non-recombinant phage. To generate libraries of P. polycephalum DNA it was necessary to digest it with suitable restriction endonucleases. P. polycephalum DNA was partially digested with either Sau3A or BamHI and the 15-25 kb fragments were isolated by electroelution. These fragments were then used to generate two genomic libraries. In each case only one type of recombinant phage was created which was derived from lambda1059 and contained a fragment of Physarum DNA. The DNA used to prepare these gene banks was found to be contaminated by a second type of DNA. This contaminating DNA was tentatively identified as mitochondrial in origin. This difficulty was eliminated when Physarum DNA was isolated by the method of Hardman & Jack (1978). DNA was partially digested with Sau3A and the 15-25 kb fragments isolated. A genomic library was prepared in lambda1059 and restriction analysis of a random sample of phage showed that all were derived from lambda1059 and all had restriction patterns different from the parental phage. Hybridization of [32P] nick-translated Physarum DNA to filter replicas of phage identified the inserts as Physarum DNA. The third part of the work involved the isolation of RNA from P. polycephalum. A requirement for screening the library was the preparation of undegraded poly(A)+ RNA from which copy DNA probes could be made. Initially an attempt was made to isolate RNA that was being actively translated on polysomes at the time of isolation. However, all attempts to prepare polysomes in sufficient quantity were unsuccessful. Cytoplasmic RNA was isolated from growing plasmodia but was highly contaminated by a polysaccharide material. This contaminant was removed by cetyltrimethylammonium bromide precipitation. Examination of the RNA, after electrophoresis under denaturing conditions showed that the RNA was very susceptible to degradation even when prepared in the presence of two inhibitors of RNase activity, RNasin and vanadyl ribonucleoside complex. Less degraded RNA was isolated in a buffer containing 4M guanidine thiocyanate, an inhibitor of RNase activity. This total RNA preparation was less degraded than the cytoplasmic RNA. When poly(A)+ RNA was isolated by oligo (dT) cellulose chromatography it directed the synthesis of very short copy DNA. The purest and most undegraded RNA was isolated by a modified version of the method described by Cox & Smulian (1983). After the initial isolation procedure the poly(A)+ RNA was further purified by a cetyltrimethylammonium bromide precipitation and a phenol/chloroform extraction. The poly(A)+ RNA was used as a template for the synthesis of cDNA in vitro which was found to be 200-900 nucleotides in length. This cDNA hybridized to filter replicas of recombinant phage. The overall conclusion from this work was that the molecular genetical techniques applied in this study have a good potential for investigating the detailed sequence of events in sporulation of Physarum polycephalum

Towards a Physarum learning chip

Networks of protoplasmic tubes of organism Physarum polycehpalum are macro-scale structures which optimally span multiple food sources to avoid repellents yet maximize coverage of attractants. When data are presented by configurations of attractants and behaviour of the slime mould is tuned by a range of repellents, the organism preforms computation. It maps given data configuration into a protoplasmic network. To discover physical means of programming the slime mould computers we explore conductivity of the protoplasmic tubes; proposing that the network connectivity of protoplasmic tubes shows pathway-dependent plasticity. To demonstrate this we encourage the slime mould to span a grid of electrodes and apply AC stimuli to the network. Learning and weighted connections within a grid of electrodes is produced using negative and positive voltage stimulation of the network at desired nodes; low frequency (10 Hz) sinusoidal (0.5 V peak-to-peak) voltage increases connectivity between stimulated electrodes while decreasing connectivity elsewhere, high frequency (1000 Hz) sinusoidal (2.5 V peak-to-peak) voltage stimulation decreases network connectivity between stimulated electrodes. We corroborate in a particle model. This phenomenon may be used for computation in the same way that neural networks process information and has the potential to shed light on the dynamics of learning and information processing in non-neural metazoan somatic cell networks