Comparative Toxicity Assessment Of Glyphosate And Two Commercial Formulations In The Planarian \u3cem\u3eDugesia japonica\u3c/em\u3e

Introduction: Glyphosate is a widely used, non-selective herbicide. Glyphosate and glyphosate-based herbicides (GBHs) are considered safe for non-target organisms and environmentally benign at currently allowed environmental exposure levels. However, their increased use in recent years has triggered questions about possible adverse outcomes due to low dose chronic exposure in animals and humans. While the toxicity of GBHs has primarily been attributed to glyphosate, other largely unstudied components of GBHs may be inherently toxic or could act synergistically with glyphosate. Thus, comparative studies of glyphosate and GBHs are needed to parse out their respective toxicity. Methods: We performed such a comparative screen using pure glyphosate and two popular GBHs at the same glyphosate acid equivalent concentrations in the freshwater planarian Dugesia japonica. This planarian has been shown to be a useful model for both ecotoxicology and neurotoxicity/developmental neurotoxicity studies. Effects on morphology and various behavioral readouts were obtained using an automated screening platform, with assessments on day 7 and day 12 of exposure. Adult and regenerating planarians were screened to allow for detection of developmentally selective effects. Results: Both GBHs were more toxic than pure glyphosate. While pure glyphosate induced lethality at 1 mM and no other effects, both GBHs induced lethality at 316 μM and sublethal behavioral effects starting at 31.6 μM in adult planarians. These data suggest that glyphosate alone is not responsible for the observed toxicity of the GBHs. Because these two GBHs also include other active ingredients, namely diquat dibromide and pelargonic acid, respectively, we tested whether these compounds were responsible for the observed effects. Screening of the equivalent concentrations of pure diquat dibromide and pure pelargonic acid revealed that the toxicity of either GBH could not be explained by the active ingredients alone. Discussion: Because all compounds induced toxicity at concentrations above allowed exposure levels, our data indicates that glyphosate/GBH exposure is not an ecotoxicological concern for D. japonica planarians. Developmentally selective effects were not observed for all compounds. Together, these data demonstrate the usefulness of high throughput screening in D. japonica planarians for assessing various types of toxicity, especially for comparative studies of several chemicals across different developmental stages

Head Removal Enhances Planarian Electrotaxis

Certain animal species utilize electric fields for communication, hunting and spatial orientation. Freshwater planarians move toward the cathode in a static electric field (cathodic electrotaxis). This planarian behavior was first described by Raymond Pearl more than a century ago. However, planarian electrotaxis has received little attention since, and the underlying mechanisms and evolutionary significance remain unknown. To close this knowledge gap, we developed an apparatus and scoring metrics for automated quantitative and mechanistic studies of planarian behavior upon exposure to a static electric field. Using this automated setup, we characterized electrotaxis in the planarian Dugesia japonica and found that this species responds to voltage instead of current, in contrast to results from previous studies using other planarian species. Surprisingly, we found differences in electrotaxis ability between small (shorter) and large (longer) planarians. To determine the cause of these differences, we took advantage of the regenerative abilities of planarians and compared electrotaxis in head, tail and trunk fragments of various lengths. We found that tail and trunk fragments electrotaxed, whereas head fragments did not, regardless of size. Based on these data, we hypothesized that signals from the head may interfere with electrotaxis when the head area/body area reached a critical threshold. In support of this hypothesis, we found that (1) smaller intact planarians that cannot electrotax have a relatively larger head-to-body-ratio than large planarians that can electrotax, and (2) the electrotaxis behavior of cut head fragments was negatively correlated with the head-to-body ratio of the fragments. Moreover, we could restore cathodic electrotaxis in head fragments via decapitation, directly demonstrating inhibition of electrotaxis by the head

Differences In Neurotoxic Outcomes Of Organophosphorus Pesticides Revealed \u3cem\u3eVia\u3c/em\u3e Multi-Dimensional Screening In Adult And Regenerating Planarians

Organophosphorus pesticides (OPs) are a chemically diverse class of commonly used insecticides. Epidemiological studies suggest that low dose chronic prenatal and infant exposures can lead to life-long neurological damage and behavioral disorders. While inhibition of acetylcholinesterase (AChE) is the shared mechanism of acute OP neurotoxicity, OP-induced developmental neurotoxicity (DNT) can occur independently and/or in the absence of significant AChE inhibition, implying that OPs affect alternative targets. Moreover, different OPs can cause different adverse outcomes, suggesting that different OPs act through different mechanisms. These findings emphasize the importance of comparative studies of OP toxicity. Freshwater planarians are an invertebrate system that uniquely allows for automated, rapid and inexpensive testing of adult and developing organisms in parallel to differentiate neurotoxicity from DNT. Effects found only in regenerating planarians would be indicative of DNT, whereas shared effects may represent neurotoxicity. We leverage this unique feature of planarians to investigate potential differential effects of OPs on the adult and developing brain by performing a comparative screen to test 7 OPs (acephate, chlorpyrifos, dichlorvos, diazinon, malathion, parathion and profenofos) across 10 concentrations in quarter-log steps. Neurotoxicity was evaluated using a wide range of quantitative morphological and behavioral readouts. AChE activity was measured using an Ellman assay. The toxicological profiles of the 7 OPs differed across the OPs and between adult and regenerating planarians. Toxicological profiles were not correlated with levels of AChE inhibition. Twenty-two “mechanistic control compounds” known to target pathways suggested in the literature to be affected by OPs (cholinergic neurotransmission, serotonin neurotransmission, endocannabinoid system, cytoskeleton, adenyl cyclase and oxidative stress) and 2 negative controls were also screened. When compared with the mechanistic control compounds, the phenotypic profiles of the different OPs separated into distinct clusters. The phenotypic profiles of adult vs. regenerating planarians exposed to the OPs clustered differently, suggesting some developmental-specific mechanisms. These results further support findings in other systems that OPs cause different adverse outcomes in the (developing) brain and build the foundation for future comparative studies focused on delineating the mechanisms of OP neurotoxicity in planarians

Comparative toxicity assessment of glyphosate and two commercial formulations in the planarian Dugesia japonica

Introduction: Glyphosate is a widely used, non-selective herbicide. Glyphosate and glyphosate-based herbicides (GBHs) are considered safe for non-target organisms and environmentally benign at currently allowed environmental exposure levels. However, their increased use in recent years has triggered questions about possible adverse outcomes due to low dose chronic exposure in animals and humans. While the toxicity of GBHs has primarily been attributed to glyphosate, other largely unstudied components of GBHs may be inherently toxic or could act synergistically with glyphosate. Thus, comparative studies of glyphosate and GBHs are needed to parse out their respective toxicity.Methods: We performed such a comparative screen using pure glyphosate and two popular GBHs at the same glyphosate acid equivalent concentrations in the freshwater planarian Dugesia japonica. This planarian has been shown to be a useful model for both ecotoxicology and neurotoxicity/developmental neurotoxicity studies. Effects on morphology and various behavioral readouts were obtained using an automated screening platform, with assessments on day 7 and day 12 of exposure. Adult and regenerating planarians were screened to allow for detection of developmentally selective effects.Results: Both GBHs were more toxic than pure glyphosate. While pure glyphosate induced lethality at 1 mM and no other effects, both GBHs induced lethality at 316 μM and sublethal behavioral effects starting at 31.6 μM in adult planarians. These data suggest that glyphosate alone is not responsible for the observed toxicity of the GBHs. Because these two GBHs also include other active ingredients, namely diquat dibromide and pelargonic acid, respectively, we tested whether these compounds were responsible for the observed effects. Screening of the equivalent concentrations of pure diquat dibromide and pure pelargonic acid revealed that the toxicity of either GBH could not be explained by the active ingredients alone.Discussion: Because all compounds induced toxicity at concentrations above allowed exposure levels, our data indicates that glyphosate/GBH exposure is not an ecotoxicological concern for D. japonica planarians. Developmentally selective effects were not observed for all compounds. Together, these data demonstrate the usefulness of high throughput screening in D. japonica planarians for assessing various types of toxicity, especially for comparative studies of several chemicals across different developmental stages

\u3cem\u3eBUMPY STEM\u3c/em\u3e Is An Arabidopsis Choline/Ethanolamine Kinase Required For Normal Development And Chilling Responses

Phospholipid biosynthesis is a core metabolic pathway that affects all aspects of plant growth and development. One of the earliest step in this pathway is mediated by choline/ethanolamine kinases (CEKs), enzymes in the Kennedy pathway that catalyze the synthesis of the polar head groups found on the most abundant plant phospholipids. The Arabidopsis genome encodes four CEKs. CEK1-3 have been well characterized using viable mutants while CEK4 encodes an essential gene, making it difficult to characterize its effects on plant development and responses to the environment. We have isolated an EMS-induced allele of CEK4 called bumpy stem (bst). bst plants are viable, allowing the effects of decreased CEK4 function to be characterized throughout the Arabidopsis life cycle. bst mutants have a range of developmental defects including ectopic stem growths at the base of their flowers, reduced fertility, and short roots and stems. They are also sensitive to cold temperatures. Supplementation with choline, phosphocholine, ethanolamine, and phosphoethanolamine rescues bst root phenotypes, highlighting the flow of metabolites between the choline and ethanolamine branches of the Kennedy pathway. The identification of bst and characterization of its phenotypes defines new roles for CEK4 that go beyond its established biochemical function as an ethanolamine kinase

Bioactivation And Detoxification Of Organophosphorus Pesticides In Freshwater Planarians Shares Similarities With Humans

Organophosphorus pesticides (OPs) are a chemically diverse class of insecticides that inhibit acetylcholinesterase (AChE). Many OPs require bioactivation to their active oxon form via cytochrome P450 to effectively inhibit AChE. OP toxicity can be mitigated by detoxification reactions performed by carboxylesterase and paraoxonase. The relative extent of bioactivation to detoxification varies among individuals and between species, leading to differential susceptibility to OP toxicity. Because of these species differences, it is imperative to characterize OP metabolism in model systems used to assess OP toxicity. We have shown that the asexual freshwater planarian Dugesia japonica is a suitable model to assess OP neurotoxicity and developmental neurotoxicity via rapid, automated testing of adult and developing organisms in parallel using morphological and behavioral endpoints. D. japonica has two cholinesterase enzymes with intermediate properties between AChE and butyrylcholinesterase that are sensitive to OP inhibition. Here, we demonstrate that D. japonica contains the major OP metabolic machinery to be a relevant model for OP neurotoxicity studies. Adult and regenerating D. japonica can bioactivate chlorpyrifos and diazinon into their respective oxons. Significant AChE inhibition was only observed after in vivo metabolic activation but not when the parent OPs were directly added to planarian homogenate using the same concentrations and timing. Using biochemical assays, we found that D. japonica has both carboxylesterase (24 nmol/(min*mg protein)) and paraoxonase (60 pmol/(min*mg protein)) activity. We show that planarian carboxylesterase activity is distinct from cholinesterase activity using benzil and tacrine. These results further support the use of D. japonica for OP toxicity studies

Acetylcholinesterase Activity Staining In Freshwater Planarians

The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system

Adult And Regenerating Planarians Respond Differentially To Chronic Drug Exposure

There is a lack of data on the effects of chronic exposure to common drugs and stimulants on the developing nervous system. Freshwater planarians have emerged as a useful invertebrate model amenable to high-throughput behavioral phenotyping to assay chemical safety in adult and developing brains. Here, we leverage the unique strength of the system to test in parallel for effects on the adult and developing nervous system, by screening ten common drugs and stimulants (forskolin, clenbuterol, LRE-1, MDL-12,330A, adenosine, caffeine, histamine, mianserin, fluoxetine and sertraline) using the asexual freshwater planarian Dugesia japonica. The compounds were tested up to 100 μM nominal concentration for their effects on planarian morphology and behavior. Quantitative phenotypic assessments were performed on days 7 and 12 of exposure using an automated screening platform. The antidepressants sertraline and fluoxetine were the most potent to induce lethality, with significant lethality observed at 10 μM. All ten compounds caused sublethal morphological and/or behavioral effects, with the most effects, in terms of potency and breadth of endpoints affected, seen with mianserin and fluoxetine. Four of the compounds (forskolin, clenbuterol, mianserin, and fluoxetine) were developmentally selective, causing effects at lower concentrations in regenerating planarians. Of these, fluoxetine showed the greatest differences between the two developmental stages, inducing many behavioral endpoints in regenerating planarians but only a few in adult planarians. While some of these behavioral effects may be due to neuroefficacy, these results substantiate the need for better evaluation of the safety of these common drugs on the developing nervous system

Let it rip: the mechanics of self-bisection in asexual planarians determines their population reproductive strategies.

Asexual freshwater planarians reproduce by transverse bisection (binary fission) into two pieces. This process produces a head and a tail, which fully regenerate within 1-2 weeks. How planarians split into two offspring-using only their musculature and substrate traction-is a challenging biomechanics problem. We found that three different species,Dugesia japonica,Girardia tigrinaandSchmidtea mediterranea, have evolved three different mechanical solutions to self-bisect. Using time lapse imaging of the fission process, we quantitatively characterize the main steps of division in the three species and extract the distinct and shared key features. Across the three species, planarians actively alter their body shape, regulate substrate traction, and use their muscles to generate tensile stresses large enough to overcome the ultimate tensile strength of the tissue. Moreover, we show thathoweach planarian species divides dictates how resources are split among its offspring. This ultimately determines offspring survival and reproductive success. Thus, heterospecific differences in the mechanics of self-bisection of individual worms explain the observed differences in the population reproductive strategies of different planarian species