29Cells, including unicellulars, are highly sensitive to external constraints from their 30 environment. Amoeboid cells change their cell shape during locomotion and in 31 response to external stimuli. Physarum polycephalum is a large multinucleated 32 amoeboid cell that extends and develops pseudopods. In this paper, changes in cell 33 behavior and shape were measured during the exploration of homogenous and non-34 homogenous environments that presented neutral, and nutritive and/or adverse 35 substances. In the first place, we developed a fully automated image analysis 36 method to measure quantitatively changes in both migration and shape. Then we 37 measured various metrics that describe the area covered, the exploration dynamics, 38 the migration rate and the slime mold shape. Our results show that: 1) Not only the 39 nature, but also the spatial distribution of chemical substances affect the exploration 40 behavior of slime molds; 2) Nutritive and adverse substances both slow down the 41 exploration and prevent the formation of pseudopods; and 3) Slime mold placed in 42 an adverse environment preferentially occupies previously explored areas rather 43 than unexplored areas using mucus secretion as a buffer. Our results also show that 44 slime molds migrate at a rate governed by the substrate up until they get within a 45 critical distance to chemical substances. 46 47 Author summary 53 54Physarum polycephalum, also called slime mold, is a giant single-celled organism 55 that can grow to cover several square meters, forming search fronts that are 56 connected to a system of intersecting veins. An original experimental protocol 57 allowed tracking the shape of slime mold placed in homogenous substrates 58 containing an attractant (glucose) or a repellent (salt), or inhomogeneous substrates 59 that contained an attractive spot (glucose), an eccentric slime mold and a repulsive 60 spot (salt) in between. For the first time, the rate of exploration of unexplored areas 61 (primary growth) and the rate of extension in previously explored areas (secondary 62 growth) were rigorously measured, by means of a sophisticated image analysis 63 program. This paper shows that the chemical composition of the substrate has more 64 influence on the morphology and growth dynamics of slime mold than that of 65 concentrated spots of chemicals. It was also found that on a repulsive substrate, 66 slime mold exhibits a bias towards secondary growth, which suggests that the mucus 67 produced during slime mold migration acts as a protective shell in adverse 68 environments. 69
70We characterize slime molds' movement both temporally and spatially, to capture the 126 6 full dynamics. To this aim, we develop a program that automatically analyzes 127 sequences of images to track the areas covered and explored by the slime mold, the 128 slime mold shape, the refinement and secondary growth cycles, as well as the 129 distance to the nutritive spot. 130 131 Results 132 1) Homogeneous environment 133In order to study the influence of the envi...