Mechanics dictate where and how freshwater planarians fission.

Asexual freshwater planarians reproduce by tearing themselves into two pieces by a process called binary fission. The resulting head and tail pieces regenerate within about a week, forming two new worms. Understanding this process of ripping oneself into two parts poses a challenging biomechanical problem. Because planarians stop "doing it" at the slightest disturbance, this remained a centuries-old puzzle. We focus on Dugesia japonica fission and show that it proceeds in three stages: a local constriction ("waist formation"), pulsation-which increases waist longitudinal stresses-and transverse rupture. We developed a linear mechanical model with a planarian represented by a thin shell. The model fully captures the pulsation dynamics leading to rupture and reproduces empirical time scales and stresses. It asserts that fission execution is a mechanical process. Furthermore, we show that the location of waist formation, and thus fission, is determined by physical constraints. Together, our results demonstrate that where and how a planarian rips itself apart during asexual reproduction can be fully explained through biomechanics.

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

Coordination of size-control, reproduction and generational memory in freshwater planarians.

Uncovering the mechanisms that control size, growth, and division rates of organisms reproducing through binary division means understanding basic principles of their life cycle. Recent work has focused on how division rates are regulated in bacteria and yeast, but this question has not yet been addressed in more complex, multicellular organisms. We have, over the course of several years, assembled a unique large-scale data set on the growth and asexual reproduction of two freshwater planarian species, Dugesia japonica and Girardia tigrina, which reproduce by transverse fission and succeeding regeneration of head and tail pieces into new planarians. We show that generation-dependent memory effects in planarian reproduction need to be taken into account to accurately capture the experimental data. To achieve this, we developed a new additive model that mixes multiple size control strategies based on planarian size, growth, and time between divisions. Our model quantifies the proportions of each strategy in the mixed dynamics, revealing the ability of the two planarian species to utilize different strategies in a coordinated manner for size control. Additionally, we found that head and tail offspring of both species employ different mechanisms to monitor and trigger their reproduction cycles. Thus, we find a diversity of strategies not only between species but between heads and tails within species. Our additive model provides two advantages over existing 2D models that fit a multivariable splitting rate function to the data for size control: firstly, it can be fit to relatively small data sets and can thus be applied to systems where available data is limited. Secondly, it enables new biological insights because it explicitly shows the contributions of different size control strategies for each offspring type.