Molecular characterization of a flatworm Girardia isolate from Guanajuato, Mexico.

Planarian flatworms are best known for their impressive regenerative capacity, yet this trait varies across species. In addition, planarians have other features that share morphology and function with the tissues of many other animals, including an outer mucociliary epithelium that drives planarian locomotion and is very similar to the epithelial linings of the human lung and oviduct. Planarians occupy a broad range of ecological habitats and are known to be sensitive to changes in their environment. Yet, despite their potential to provide valuable insight to many different fields, very few planarian species have been developed as laboratory models for mechanism-based research. Here we describe a previously undocumented planarian isolate, Girardia sp. (Guanajuato). After collecting this isolate from a freshwater habitat in central Mexico, we characterized it at the morphological, cellular, and molecular level. We show that Girardia sp. (Guanajuato) not only shares features with animals in the Girardia genus but also possesses traits that appear unique to this isolate. By thoroughly characterizing this new planarian isolate, our work facilitates future comparisons to other flatworms and further molecular dissection of the unique and physiologically-relevant traits observed in this Girardia sp. (Guanajuato) isolate.

The effect of stress on rates of asexual reproduction in an invasive planarian.

Animal reproduction under stressful conditions is often reduced, with current survival and future reproduction being generally traded off against current reproductive activity. This study examines the impacts of physical and chemical stressors on the rates of asexual reproduction of the invasive planarian Girardia tigrina. 320 wild-caught planaria (mixed size class) were kept individually in Petri dishes such that their individual rates of fission through fragmentation could be easily monitored. Four treatment groups were compared, one chemical (5 mg/L ammonia) and one physical (decapitation), in comparison to a negative control (animals were starved of food) and a positive control where the animals were given an abundance of food. The two treatment groups immediately began reproducing asexually and accumulated the highest number of fissions over the course of the 12-day investigation period, while the positive control only began to fission after 7 days. We propose that the reproductive response observed here is an adaptive one to stressful conditions, whereby the likelihood of survival through numerical abundance is enhanced, although the size and vulnerability of resulting fragments may impose a balancing cost. The response may play a role in the invasiveness of G. tigrina by making it able to colonize environments where adverse conditions prevail.

Oxygen consumption rate of flatworms under the influence of wake- and sleep-promoting neurotransmitters.

Flatworms are among the best studied animal models for regeneration; however, they also represent an emerging opportunity to investigate other biological processes as well. For instance, flatworms are nocturnal and sleep during the day, a state that is regulated by sleep/wake history and the action of the sleep-promoting neurotransmitter gamma-aminobutyric acid (or GABA). Sleep is widespread across the animal kingdom, where it serves many nonexclusive functions. Notably, sleep saves energy by reducing metabolic rate and by not doing something more energetically taxing. Whether the conservation of energy is apparent in sleeping flatworms is unclear. We measured the oxygen consumption rate (OCR) of flatworms dosed with either (1) GABA (n = 29) which makes flatworms inactive or (2) dopamine (n = 20) which stimulates flatworms to move, or (3) day and night neurotransmitter-free controls (n = 28 and 27, respectively). While OCR did not differ between the day and night, flatworms treated with GABA used less oxygen than those treated with dopamine, and less than the day-time control. Thus, GABA affected flatworm physiology, ostensibly by enforcing energy-conserving sleep. Evidence that dopamine increased metabolism was less strong. This work broadens our understanding of flatworm physiology and expands the phylogenetic applicability of energy conservation as a function of sleep.

Persistence of Nocturnality in Decapitated and Bisected Flatworms.

The ability of flatworms to regenerate entire brain structures, and indeed much of their body from mere fragments of the whole animal, presents the unique opportunity to observe the development of day-night rhythms in adult animals. In many animals, young are arrhythmic, and their species-specific timing of activity develops as the animal matures. In this study, we created two flatworm cohorts, housed in isolation, that were regenerating either (1) the brain in a decapitated animal, or (2) major body structures in a bisected, tailless animal. In this way, we observed how bisection influenced the level of activity and diel rhythmicity, and how these developed as each flatworm regenerated. Here, we demonstrate that intact flatworms were predominantly active at night, with peaks in activity seen in the hours after lights-off and before lights-on. While decapitated and tailless flatworms could still move, both were less active than the original animal, and both segments retained a nocturnal lifestyle. Furthermore, decapitated flatworms, once regenerated, again showed a U-shaped pattern of nocturnal activity reminiscent of the two night-time peaks seen in the original animal. These results could be used to further investigate how regeneration may affect motor control and motor output, or to further investigate the presence of a clock in the flatworm brain.

Why study sleep in flatworms?

The behaviors that characterize sleep have been observed across a broad range of different species. While much attention has been placed on vertebrates (mostly mammals and birds), the grand diversity of invertebrates has gone largely unexplored. Here, we introduce the intrigue and special value in the study of sleeping platyhelminth flatworms. Flatworms are closely related to annelids and mollusks, and yet are comparatively simple. They lack a circulatory system, respiratory system, endocrine glands, a coelom, and an anus. They retain a central and peripheral nervous system, various sensory systems, and an ability to learn. Flatworms sleep, like other animals, a state which is regulated by prior sleep/wake history and by the neurotransmitter GABA. Furthermore, they possess a remarkable ability to regenerate from a mere fragment of the original animal. The regenerative capabilities of flatworms make them a unique bilaterally symmetric animal to study a link between sleep and neurodevelopment. Lastly, the recent applications of tools for probing the flatworm genome, metabolism, and brain activity make their entrance into the field of sleep research all the more timely.

Neurotransmitters of sleep and wakefulness in flatworms.

STUDY OBJECTIVES: Sleep is a prominent behavioral and biochemical state observed in all animals studied, including platyhelminth flatworms. Investigations into the biochemical mechanisms associated with sleep-and wakefulness-are important for understanding how these states are regulated and how that regulation changed with the evolution of new types of animals. Unfortunately, beyond a handful of vertebrates, such studies on invertebrates are rare. METHODS: We investigated the effect of seven neurotransmitters, and one pharmacological compound, that modulate either sleep or wakefulness in mammals, on flatworms (Girardia tigrina). Flatworms were exposed via ingestion and diffusion to four neurotransmitters that promote wakefulness in vertebrates (acetylcholine, dopamine, glutamate, histamine), and three that induce sleep (adenosine, GABA, serotonin) along with the H1 histamine receptor antagonist pyrilamine. Compounds were administered over concentrations spanning three to five orders of magnitude. Flatworms were then transferred to fresh water and video recorded for analysis. RESULTS: Dopamine and histamine decreased the time spent inactive and increased distance traveled, consistent with their wake-promoting effect in vertebrates and fruit flies; pyrilamine increased restfulness and GABA showed a nonsignificant trend towards promoting restfulness in a dose-dependent manner, in agreement with their sleep-inducing effect in vertebrates, fruit flies, and Hydra. Similar to Hydra, acetylcholine, glutamate, and serotonin, but also adenosine, had no apparent effect on flatworm behavior. CONCLUSIONS: These data demonstrate the potential of neurotransmitters to regulate sleep and wakefulness in flatworms and highlight the conserved action of some neurotransmitters across species.

Do bio-insecticides affect only insect species? Behavior, regeneration, and sexual reproduction of a non-target freshwater planarian.

Bio-insecticides have been increasingly used worldwide as ecofriendly alternatives to pesticides, but data on their effects in non-target freshwater organisms is still scarce and limited to insects. The aim of this study was to determine the lethal and sub-lethal effects of the bio-insecticides Bac Control (based on Bacillus thuringiensis kurstaki-Btk) and Boveril (based on Beauveria bassiana-Bb) on regeneration, behavioral, and reproductive endpoints of the freshwater planarian Girardia tigrina. The estimated LC50-48h were > 800 mg a.i./L for Btk and 60.74 mg a.i./L for Bb. In addition, exposure to Btk significantly decreased locomotion and feeding activities of planarians (lowest observed effect concentration (LOEC) of 12.5 mg a.i./L Btk) and fecundity rate (LOEC = 3.12 mg a.i./L Btk), whereas exposure to Bb significantly delayed regeneration (LOEC = 0.75 mg a.i./L Bb) and decreased fecundity rate (1.5 mg a.i./L Bb) of planarians. Thus, both bio-insecticides induced deleterious sub-lethal effects on a non-insect freshwater invertebrate species. However, only Bb-based formulation affected the survival, fecundity rate, and regeneration at concentrations below the maximum predicted environmental concentration (PEC = 247 mg/L). Thus, care should be taken when using such formulations as alternatives to chemical insecticides near aquatic ecosystems.

The complete mitochondrial genome of a planarian flatworm Girardia tigrina (Tricladida: Dugesiidae).

Girardia tigrina, a freshwater planarian species native to America and introduced to other continents, has been usually used as model organism in many research fields of biology. In this study, we determined the complete mitochondrial genome of G. tigrina using next-generation sequencing (NGS). The complete mitogenome was 15,938 bp in length, with 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs, and ATP8 was absent in the mitogenome of G. tigrina as in the mitogenomes of some other flatworms. The maximum-likelihood phylogenetic tree suggested that G. tigrina was closely related to genus Dugesia in the clade of Tricladida.

Regeneration of Planarian Auricles and Reestablishment of Chemotactic Ability.

Detection of chemical stimuli is crucial for living systems and also contributes to quality of life in humans. Since loss of olfaction becomes more prevalent with aging, longer life expectancies have fueled interest in understanding the molecular mechanisms behind the development and maintenance of chemical sensing. Planarian flatworms possess an unsurpassed ability for stem cell-driven regeneration that allows them to restore any damaged or removed part of their bodies. This includes anteriorly-positioned lateral flaps known as auricles, which have long been thought to play a central role in chemotaxis. The contribution of auricles to the detection of positive chemical stimuli was tested in this study using Girardia dorotocephala, a North American planarian species known for its morphologically prominent auricles. Behavioral experiments staged under laboratory conditions revealed that removal of auricles by amputation leads to a significant decrease in the ability of planarians to find food. However, full chemotactic capacity is observed as early as 2 days post-amputation, which is days prior from restoration of auricle morphology, but correlative with accumulation of ciliated cells in the position of auricle regeneration. Planarians subjected to x-ray irradiation prior to auricle amputation were unable to restore auricle morphology, but were still able to restore chemotactic capacity. These results indicate that although regeneration of auricle morphology requires stem cells, some restoration of chemotactic ability can still be achieved in the absence of normal auricle morphology, corroborating with the initial observation that chemotactic success is reestablished 2-days post-amputation in our assays. Transcriptome profiles of excised auricles were obtained to facilitate molecular characterization of these structures, as well as the identification of genes that contribute to chemotaxis and auricle development. A significant overlap was found between genes with preferential expression in auricles of G. dorotocephala and genes with reduced expression upon SoxB1 knockdown in Schmidtea mediterranea, suggesting that SoxB1 has a conserved role in regulating auricle development and function. Models that distinguish between possible contributions to chemotactic behavior obtained from cellular composition, as compared to anatomical morphology of the auricles, are discussed.

Laboratory Maintenance and Propagation of Freshwater Planarians.

Freshwater planarians are a powerful model organism for the study of animal regeneration, stem cell maintenance and differentiation, and the development and functions of several highly conserved complex tissues. At the same time, planarians are easy to maintain, inexpensive to propagate, and reasonably macroscopic (1 mm to 1 cm in length), making them excellent organisms to use in both complex academic research and hands-on teaching laboratories. Here, we provide a detailed description of how to maintain and propagate these incredibly versatile animals in any basic laboratory setting. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Salt solution preparation Basic Protocol 2: Cleaning planarian housing Basic Protocol 3: Food preparation Basic Protocol 4: Feeding planarians Basic Protocol 5: Expansion and amplification of colony.

Analysis of beta-blocker bioconcentration in brown planaria (Girardia dorotocephala) and its effects on regeneration.

Production, distribution, and disposal of pharmaceutical products, including beta-blockers, have become a global issue. Beta-blockers are known to persist in the environment months after their release and may result in the disruption of the homeostatic system in non-target organisms. Here, we study the bioconcentration of three of the most commonly used beta-blockers and their effect on the regeneration of Girardia dorotocephala, a freshwater brown planarian. Acute toxicity tests determined LC50s for acebutolol, metoprolol, and propranolol to be 778 mg/L, 711 mg/L, and 111 mg/L, respectively. The quantification and analysis of beta-blocker bioconcentration during acute exposure were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After 4 days of exposure to beta-blockers, the bioconcentration drastically decreased for all three beta-blockers at all exposure levels, suggesting that an effective mechanism to reduce uptake or excrete beta-blockers could be present. Additionally, Girardia dorotocephala were cut proximal to the head and the quality of regeneration was documented from each fragment daily. No significant difference was visually observed after 2 weeks of regeneration between the brown planarians placed in beta-blocker solution and those placed in control solution.

Exposure to Roundup® affects behaviour, head regeneration and reproduction of the freshwater planarian Girardia tigrina.

The demand of glyphosate-based herbicides including Roundup® is rising in the tropics due to increase occurence of glyphosate-resistant weeds that require higher herbicide application rates but also because of their use associated with genetically engineered, glyphosate-tolerant crops. Consequently, there is now an excessive use of glyphosate in agricultural areas with potential adverse effects also for the surrounding aquatic environments. This study aimed to determine the sensitivity of the freshwater planarian Girardia tigrina to acute and chronic exposures of Roundup®. Planarians were exposed to a range of lethal and sub-lethal concentrations of Roundup® to determine the median lethal concentration (LC50) concerning its active ingredient glyphosate and also effects on locomotor velocity (pLMV), feeding rate, regeneration, reproductive parameters and morphological abnormalities. Regeneration endpoints included length of blastema and time for photoreceptors and auricles regeneration after decapitation, while effects on reproduction were assessed measuring fecundity (number of deposited cocoons) and fertility (number of hatchlings) over five weeks of exposure to glyphosate. The estimated 48 h LC50 of was 35.94 mg glyphosate/L. Dose dependent effects were observed for feeding, locomotion and regeneration endpoints with Lowest observed effect concentration (LOEC) values as low as 3.75 mg glyphosate/L. Chronic exposures to environmentally relevant concentrations of glyphosate significantly impaired fecundity and fertility rates of exposed planarians (median effective concentration, EC50 = 1.6 mg glyphosate/L for fecundity and fertility rates). Our results show deleterious effects of Roundup® on regeneration, behavior and reproduction of freshwater planarians and add important ecotoxicological data towards the environmental risk assessment of glyphosate-based herbicide in freshwater ecosystems.

Two new sympatric troglobitic freshwater flatworms (Platyhelminthes: Dugesiidae) from a hotspot of subterranean biodiversity in the Neotropics.

The Areias System, situated in the Atlantic Forest, is considered a hotspot of troglobitic diversity in Brazil. Herein we describe two new sympatric, troglobitic species of Girardia occurring in this system. Both species show minute eyes; one of them has a whitish body, and the other scattered, fine pigmentation over the dorsal surface. Regarding the copulatory apparatus, one of the species shows a bulbar cavity with multiple diverticula and a bursal canal of the angled type, whereas the other has a single, ovoid bulbar cavity and a smoothly curved bursal canal, among other distinguishing features. Thus, each species can be differentiated by a unique combination of features in its external morphology and copulatory apparatus. Both present a restricted distribution, occurring in a group of pools in travertine rock formed by water from the epikarst. The morphological features of both species and this sampling location indicate that both species are stygobionts.

Inactivity Is Nycthemeral, Endogenously Generated, Homeostatically Regulated, and Melatonin Modulated in a Free-Living Platyhelminth Flatworm.

Introduction: Sleep either appeared once early in the evolution of animals, or at multiple instances over evolutionary time. Understanding whether sleep is a diagnostic trait for members of the kingdom Animalia has important implications for our understanding of the evolution of sleep and sleep functions. Unfortunately, knowledge on the phylogenetic breadth of sleep is restricted to vertebrates, a few arthropods and molluscs, and one species of nematode. There is a dearth of information on the other 30 or so animal phyla. Aims and Methods: Here, we provide original data on a previously unstudied group of animals with respect to sleep: platyhelminth flatworms. These free-living animals are relatively simple, with a rudimentary central nervous system and absence of many other specialized physiological systems. Results: Despite this simplicity, inactive flatworms appeared to be sleeping. Specifically, quiescence was organized in a circadian manner, occurring largely during the daytime. This basic rhythm persisted under constant darkness, suggesting that it was endogenously generated. Active flatworms responded more readily to stimulation, and flatworms recovered lost sleep by sleeping longer after a 3-hour period of inactivity deprivation. We were also able to increase inactivity in a dose-dependent manner with exposure to melatonin, a hormone that increases sleep in diurnal animals. Conclusions: Taken together, these data expand our understanding of the phylogenetic extent of sleep and reinforce the idea that sleep evolved early in the evolutionary history of animals. However, additional studies on other types of animals are required for a comprehensive understanding of the origin(s) and evolution of sleep.

Effects of a textile azo dye on mortality, regeneration, and reproductive performance of the planarian, Girardia tigrina.

BACKGROUND: Many dyes can be considered emerging contaminants. The most widely used dyes belong to the class of azo compounds, some of which are known to have toxic and genotoxic properties. They are used in great quantities in textile activities and are of environmental concern because of their potential discharge in water. Planarians have been successfully used as test organisms in hazard evaluation of different chemicals, and we demonstrate the suitability of Girardia tigrina for laboratory testing. The aim of this work was to evaluate the suitability of the planarian species G. tigrina to assess the ability of the azo textile dye disperse red 1 to cause acute toxicity and adverse effects in the regeneration and reproduction of newborn and adult specimens. RESULTS: Disperse red 1 presented a median LC50 of 75 and 152 mg/L, respectively, for newborns and adults of G. tigrina, showing that newborns are twice as susceptible to the dye. Uncoordinated movements, irregular twists, colored skin, increased mucous production, and regenerative delays were observed after dye exposure at sub-lethal concentrations. CONCLUSIONS: A no-observed-adverse-effect concentration (NOAEC) of 0.1 mg/L could be determined for disperse red 1 based on the fecundity test. Zinc seems to be a suitable positive control for monitoring the sensitivity in G. tigrina tests within only 24 h of exposure. This study demonstrates the applicability of G. tigrina tests in the hazard evaluation of water contaminants, such as azo dyes.

A study of low power laser on the regenerative process of Girardia tigrina (Girard, 1850) (Turbellaria; Tricladida; Dugesiidae) / Um estudo de laser de baixa potência no processo regenerativo de Girardia tigrina (Girard, 1850) (Turbellaria; Tricladida; Dugesiidae)

The mechanism of regeneration does not start to restore the wound until its corresponding epimorphic phase. A bioestimulation of tissues and cells by laser radiation depends on the wavelength, on the dose, and on the intensity of the light. The goal of this work was to verify the effect of the low power laser at 660 nm on the regenerative process of Girardia tigrina. The specimens were maintained in the laboratory under a temperature ranging from 19° up to 24 °C for 21 days. The planarians were anesthetized by placing them on ice and then cut them with a scalpel. The three treatments were as following: animals individually irradiated with 14 sessions with 1 minute duration (treatment 1), 14 sessions with 3 minutes duration (treatment 2), and without irradiation (control). The planarians were amputated and divided in three study treatments: a control group (without radiation), and two other treatments: irradiated for 1 minute, and irradiated for 3 minutes. The animals were irradiated with diode laser (660 nm) with 3.3 ± 0.3 mW of power, using 0.94 mW.mm-2 power density for each irradiation procedure. During the experiment, 14 irradiation sessions were undertaken. The specimens were fixed in Bouin, and stained with hematoxyline and eosin. From observation and histological analysis, it was possible to assess the effects of interaction between laser and tissue. The head fragment after 1 minute of irradiation presented a better organized tissue scheme, when compared with the other treatments. Aspects of the body fragments submitted to 3 minutes of light treatment were very similar to fragments that had not been injured. It can be concluded that there are changes in the quality of regeneration when treated with low power laser under the conditions mentioned above.

O mecanismo de regeneração não restaura a ferida até sua fase epimórfica correspondente. A bioestimulação de tecidos e células por radiação laser depende do comprimento de onda, da dose e intensidade de luz. O objetivo deste trabalho foi verificar o efeito do laser de baixa potência (660 nm) no processo regenerativo de Girardia tigrina. Estes vermes têm uma elevada capacidade de regeneração. Os espécimes foram mantidos em laboratório sob uma temperatura de 19° a 24 °C por 21 dias. As planárias foram anestesiadas com gelo e amputadas com bisturi. Os 3 tratamentos foram compostos de: animais individualmente irradiados com 14 sessões de 1 minuto de duração (tratamento 1), com 14 sessões de três minutos de duração (tratamento 2) e não irradiados (controle). Elas foram irradiadas com laser diodo (660 nm) 3,3 ± 0,3 mW potência, usando 0.94 mW.mm-2 de densidade de potência para cada irradiação. Durante o experimento foram realizadas 14 sessões. Os espécimes foram fixados em Bouin e corados com hematoxilina e eosina. Com base nas observações e análise histológica, foi possível avaliar os efeitos da interação entre laser e tecido. O fragmento cabeça após 1 minuto de irradiação mostrou um tecido melhor organizado quando comparado com outros tratamentos. Os aspectos dos fragmentos corpo após 3 minutos de irradiação foram muito similares para os fragmentos que não sofreram injúria. Existem mudanças na qualidade de regeneração quando tratadas com laser nas condições acima mencionadas.