Toxicity profiles and solvent–toxicant interference in the planarian Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure

To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound‐induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent–toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short‐ (1 day) and long‐term (2 weeks) exposures to low DMSO concentrations (up to 1000 µl l^?1) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long‐term exposure set‐up, a concentration of 500 µl l^?1 DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 µl l^?1 DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 µl l^?1 DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 µl l^?1 in this organism. In the second part of our study, we propose a statistical approach to account for solvent–toxicant interactions and discuss full‐scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea. Copyright © 2014 John Wiley & Sons, Ltd.     

From the Cover: Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye–brain-independent movement and arousal

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye–brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin–expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in “adult-like animals.” Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

Light sensing has independently evolved multiple times and has profoundly shaped life. The ability to process light information in distinct ways and respond to a changing light environment can dramatically shape physiology and fitness of life forms. Movement, triggered by light, is one of the most fundamental responses in nature (1). Among metazoans, a wide variety of animals are known to show coordinated motion in response to light stimuli. So far, this is overwhelmingly known to be mediated through the animal eyes. In fact, eye-driven light sensing and taxis has been extensively studied across phyla. Interestingly, motion in metazoans can also be mediated through eye-independent or extraocular (EO) photoreception (2–5). However, our conceptual and mechanistic grasp on how coordinated movement can be triggered and controlled through EO light-sensing systems is extremely limited. Moreover, the few prominent examples of EO phototaxis have all been reported in life forms/developmental stages completely lacking eyes or possessing only rudimentary eyes (2, 5). Almost nothing is known about sensitive EO light-sensing systems capable of triggering coordinated motion that may coexist with sensitive eyes in a single organism.There are intriguing reports of photoreceptor molecules that are expressed in locations other than conventional eyes, including in unusual structures seen in polyclad flatworms, clitellate segmented worms, crustaceans, cephalopods, and fishes. However, the functions of such structures remain elusive (6–13). A single organism may indeed possess multiple, independent light-responsive systems, both eye based as well as eye independent (13–17), but the functions rarely overlap. “Nonvisual”/EO sensory systems like pineal glands and deep-brain photoreceptors across vertebrates and retinal ganglion cells in mammals (18–21) have been reported. However, these sensory systems are generally known to perform “nonvisual” functions like maintaining circadian rhythms and modulating behavior (22–25). Here, we report an EO phototactic network that can independently trigger coordinated movement just like what the eye-based (ocular) system can, while also having its own distinctive role even when the eyes are present.Do highly sensitive EO phototactic systems coexist and function in life forms that have prominent eye-based networks as well? How would such a system operate? What would be the mechanistic framework and the functional consequence of such an eye-independent light-sensory system? Planarian flatworms offer a fascinating opportunity to explore such a paradigm. Planarians are highly light aversive and have well-developed ocular cerebral eyes (eyes connected to a centralized ganglion) that process light stimuli and guide behavior like feeding, escape, and predation (26–30). In fact, the planarian ocular network is highly sensitive and capable of surprisingly complex processing (17). These eye-mediated behaviors are reliant on an organized, cephalized bilobed brain, a prominent example of a “primitive” brain in evolution (17, 31, 32). Indeed, the brain is required for most locomotive behaviors including thermotaxis, chemotaxis, and eye-mediated phototaxis including the ability to discriminate closely related light stimuli, shown by these animals (17, 32–34). However, planarians also show dramatic, eye–brain-independent light-induced movements (17, 35). Even after sudden decapitation (removal of both eyes and brain), worms are able to acutely respond to ultraviolet-A (UV-A) light and show seemingly coordinated movement away from light (17, 35). While such eye–brain-independent behavior has long fascinated biologists, almost nothing is known about how this dramatic behavior is mediated (17, 35–39). It is also not clear what would be the physiological role of such an acutely sensitive EO sensory network capable of triggering coordinated movement, especially since planarians do have a well-developed ocular network.Here, we show how such an acute response to light is mediated by an organism removed of its “primary” light-sensory organ and brain. We report the discovery of photoreceptor molecules as well as a widespread but organized network of photoreceptor cells that are required for this acute eye-independent UV-A light response. Intriguingly, this entire multiscale sensory system from photoreceptors to the network of cells arises and matures postembryonically, in an “adult-like” organism. This developmental trajectory is distinct from that of the cerebral eyes, which develop embryonically. We also demonstrate that while both the eyes as well the EO network led to coordinated movements relying on the same locomotion machinery, the physiological consequences of the ocular and EO sensory responses can be divergent. Intact planarians periodically go into “sleep-like” resting phases, in which their activity diminishes and sensory perception reduces (40). Strikingly, we find that the EO sensory system in these intact animals can override the natural “rest”-activity cycles and is able to acutely photoactivate and arouse even resting worms. This is distinct from the ocular network that becomes dormant during the “rest phase.” Our work illustrates an unprecedented level of organization and complexity in form and function of an acutely sensitive EO light-sensory system that matures and functions in parallel to the ocular network.     

Toxicity of nitrite to three species of freshwater invertebrates

Nitrite is a compound with a high toxicity to aquatic animals. Several anthropogenic pollution sources are increasing the concentrations of this component of the nitrogen cycle. Despite this toxicity, there is little available literature on its effects on freshwater invertebrates. Laboratory bioassays were performed to obtain data on the lethal effects of nitrite to three species of freshwater invertebrates: the planarian Polycelis felina and the amphipods Echinogammarus echinosetosus and Eulimnogammarus toletanus. The LC(50), LC(10), and LC(0.01) values (mg/L NO(2)--N) at 24, 48, 72, and 96 h were calculated for each species. E. toletanus and E. echinosetosus were the most sensitive species, with 96 h LC(50) values of 2.09 and 2.59 mg/L NO(2)--N, respectively. In contrast, the planarian P. felina showed a higher tolerance to nitrite, with a 96 h LC(50) value of 60.0 mg/L NO(2)--N. The obtained results were compared with the reported nitrite data for other freshwater invertebrates. This study may contribute to a more appropriate assessment of the ecological risk of this compound in freshwater ecosystems.     

Freshwater planarians as novel organisms for genotoxicity testing: Analysis of chromosome aberrations

Two freshwater species of planarians, Girardia schubarti Marcus and G. tigrina Girard, were used for measuring chromosome aberration (CA) induction under laboratory conditions. Three genotoxicants were tested: methyl methanesulfonate (MMS), a direct-acting genotoxicant; cyclophosphamide, a metabolism-dependent genotoxicant; and gamma-radiation, a clastogenic agent. All three agents produced positive responses in both species. The strongest dose-responses were detected with MMS, and, in general, G. tigrina was somewhat more sensitive to the genotoxicity of the agents than G. schubarti. This difference in sensitivity may be due to: (a) the smaller body mass of G. tigrina; (b) differences in DNA repair, which may be reflected in the marginally higher background CA frequency of G. tigrina; and/or (c) the greater number of chromosomes in G. tigrina (2N = 16) as compared with G. schubarti (2N = 8). The responses induced by gamma-radiation in the planarians were similar to or higher than those induced in cultured human lymphocytes. The CA-planarian assay has advantages for monitoring environmental genotoxicity in natural water resources or urban and industrial wastewater since planarians are characterized by (a) a relatively low number of easily analyzable chromosomes; (b) high regenerating capacity, allowing exposure of replicating cells from different parts of the same organism to different doses; (c) easy maintenance under laboratory conditions; and (d) worldwide distribution, making them available for genotoxicity tests using either in situ or controlled laboratory exposure conditions.     

A Lissencephaly‐1 homologue is essential for mitotic progression in the planarian Schmidtea mediterranea

COVER PHOTOGRAPH: Pseudocolored low magnification image of the planarian Schmidtea mediterranea immunostained using (in top right image) anti‐β‐tubulin (white), anti‐phosphorylated histone H3 (green) and DAPI (magenta), which label the central nervous system, mitotic cells and nuclei, respectively. Center image was captured using DIC. At higher magnification, these stains were used to analyze the mitotic spindle apparatus of dividing cells. From Cowles et al., Developmental Dynamics 241:901–910, 2012.     

A Lissencephaly-1 homologue is essential for mitotic progression in the planarian Schmidtea mediterranea

Background : Planarians are renowned for their capacity to replace lost tissues from adult pluripotent stem cells (neoblasts). Here we report that Lissencephaly‐1 (lis1), which has roles in cellular processes such as mitotic spindle apparatus orientation and in signal regulation required for stem cell self‐renewal, is required for stem cell maintenance in the planarian Schmidtea mediterranea. Results : In planarians, lis1 is expressed in differentiated tissues and stem cells. lis1 RNAi leads to head regression, ventral curling, and death by lysis. By labeling the neoblasts and proliferating cells, we found lis1 knockdown animals show a dramatic increase in the number of mitotic cells, followed by depletion of the stem cell pool. Analysis of the mitotic spindles in dividing neoblasts revealed that defective spindle positioning is correlated with cells arrested at metaphase. In addition, we show that inhibiting a planarian homologue of nudE, predicted to encode a LIS‐1 interacting protein, also leads to cell cycle progression defects. Conclusions : Our results provide evidence for a conserved role of LIS1 and NUDE in regulating the function of the mitotic spindle apparatus in a representative Lophotrochozoan and that planarians will be useful organisms in which to investigate LIS1 regulation of signaling events underlying stem cell self‐renewal. Developmental Dynamics 241:901–910, 2012. © 2012 Wiley Periodicals, Inc.