Regeneration of photoreceptor organs in freshwater planarians at different levels of accumulation of natural methylmercury compounds

The effects of natural methylmercury compounds on regeneration of photoreceptor organs were studied in three freshwater planarians: Polycelis tenuis, Dugesia lugubris, and D. tigrina. Accumulation of methyl mercury in the planarian body suppressed regeneration of P. tenuis with numerous photoreceptor organs to a greater extent than in two other planarians that have only two eyes. High methyl mercury concentrations inhibited the restoration of photoreceptor organs in asexual and sexual D. tigrina races.     

Regeneration of freshwater planarians <Emphasis Type="Italic">Dugesia tigrina</Emphasis> and <Emphasis Type="Italic">Polycelis tenuis</Emphasis> under the influence of methyl mercury compounds of natural origin

We studied the effects of methyl mercury compounds of natural origin on regeneration of the planarians Dugesia tigrina and Polycelis tenuis. Accumulation of methyl mercury in the planarian body leads to a delayed formation of photoreceptor organs in planarians of both species. After a significant traumatic load, the regeneration is suppressed and the death of some control and most experimental animals was observed. The intensity of joining additional cuts depends on the localization of body fragment with a cut and localization of a cut itself.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 35–40.Original Russian Text Copyright © 2005 by Medvedev, Komov.     

Regeneration of freshwater planarians Dugesia tigrina and Polycelis tenuis under the influence of methyl mercury compounds of natural origin

We studied the effects of methyl mercury compounds of natural origin on regeneration of the planarians Dugesia tigrina and Polycelis tenuis. Accumulation of methyl mercury in the planarian body leads to a delayed formation of photoreceptor organs in planarians of both species. After a significant traumatic load, the regeneration is suppressed and the death of some control and most experimental animals was observed. The intensity of joining additional cuts depends on the localization of body fragment with a cut and localization of a cut itself.     

An immunocytochemical method for histamine: application to the planarians

Histamine-immunoreactivity was investigated in the planarians Dugesia tigrina and Polycelis nigra. Specific antisera against a histamine-protein conjugate were used, and 1-ethyl—3 (3-dimethyl-aminopropyl) carbodiimide was used both as coupling agent to prepare the antigen and as a tissue fixative. In D. tigrina, histamine-immunoreactivity was restricted to photoreceptor cells in the cerebral eye. In P. nigra, nerve fibers were found in the ventral nerve cord and nerves running laterally from these. The epidermal eyes did not display histamine-immunoreactivity. The results suggest that histamine may be a transmitter in some of the most primitive animals. They also suggest that the distribution of histamine may differ in planarians.     

Specific features of eye regeneration in multiocular planarians <Emphasis Type="Italic">Polycelis tenuis</Emphasis>

Regeneration and negative phototaxis were studied in planarians Polycelis tenuis, in which the anterior body end is fringed with many eyes. Comparative data for the same indices are given for binocular planarians Girardia tigrina. Multiple eyes regenerated gradually with a decrease in the rate of regeneration and independently from the rate of restoration of the anterior body end, where they are located. Negative phototaxis was restored independently from the total amount of regenerated eyes. It was unstable in both planarian species.     

Peptidergic signalization: An immunocytochemical study of FMRF-related peptides in the nervous system of planarians

Free-living freshwater flatworms, planarians, are well known biological object. Due to their unprecedented regenerative ability, planarians attract the particular attention of scientists. From a small body piece planarians can regenerate a whole organism including central nervous system and all organs and tissues. Employment of modern immunocytochemical methods in conjunctions with confocal laser scanning microscopy (CLSM) made it possible to discover a spectrum of neuronal substances in the nervous system of relatively simple animals. In the present study, specific fluorescently labeled antibodies have been used for identification of FMRF-related neuropeptides in the nervous system of three planarian species: Schmidtea mediterranea, Girardia tigrina, and Polycelis tenuis (Platyhelminthes, Turbellaria). The details of the FMRF-like immunostaining have been described and completed. Some previously published data concerning neuronal signalization in turbellarians have been observed.     

Some details on the morphological structure of planarian musculature identified by fluorescent and confocal laser-scanning microscopy

The details of the morphological organization of the body musculature in the planarians Girardia tigrina and Polycelis tenuis were investigated by histochemical staining of actin filaments with fluorescently labeled fluorescent. The whole mount preparations and frozen tissue sections of planarians were analyzed by fluorescent and confocal laser scanning microscopy. The results indicate that the muscle system is well differentiated in both planarian species and is represented by the somatic musculature of the body wall, the musculature of the digestive tract, and the musculature of the reproductive system organs in P. tenuis, which reproduces sexually. The differences and similarities between the two species in the morphological characters of the musculature, which are the size and density of myofibrils in different muscle layers, were described. The results present the basis for further studies on the regulation of muscle function in planarians.     

Retinoic acid as a regulator of planarian morphogenesis

The effect of retinoic acid on regeneration of two species of asexual planarian races, Girardia tigrina and Schmidtea mediterranea, was studied. It was established that retinoic acids at physiological concentrations (10⁻⁷–10⁻¹⁰ M) inhibit the regeneration of the head part of planarians but have no effect on tail blastema growth. It is shown that regeneration of the head part is inhibited as a result of arrest of the cell cycle of neoblasts, proliferating stem cells, during the transition from the G ₁/G ₀ to the S phase. Thus, the morphogenetic role of retinoic acids in planarians, primitive bilaterally symmetrical animals, has been demonstrated.     

Formation of the function of the photosensing system in early development

The function of simple eyes in two planarian species, two-eyed Girardia tigrina and multi-eyed Polycelis tenuis, has been studied. When exposed to light, planarians display a light avoidance reaction known as negative phototaxis. This reaction has been investigated in intact animals and in head and tail fragments in the course of eye regeneration after their section. Specific features of the phototaxis reaction have been described in all groups of animals. The differences in light response recovery were shown between two planarian species and two regenerating fragments. No correlation has been found between phototactic reactions and restoration of eye structure, the number of eyes, maturation of the ganglion, growth of regenerative blastema, and motor system. The phototactic response occurred two days after the recovery of the morphology of eyes and their connection with the brain. The participation of conserved and novel genes in early development of the eye function is discussed.     

Asexual reproduction of planarians: Metric studies

A relationship was studied between fission and restoration of body and its individual parts under different experimental conditions in planarians of the Dugesia tigrina asexual race. The body and its fragments were studied morphomterically. After fission, the growth of planarians demonstrated topographic differences. The separated tail fragments and postpharyngeal area, in which the zone of fission is formed, were growing at the highest rate. More active growth was also noted over the long body axis. Fission and growth were more active in isolated planarians, as compared to those kept in groups.     

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

 To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians. Accepted: 16 September 1996     

Weak influences of physical and chemical factors on the morphogenetic process (in Invertebrates)

Analysis is presented of long-term data obtained in investigation of the effects of weak influences on morphogenetic processes in invertebrates (regeneration of planarians Dugesia (Girardia) tigrina and postembryonic development of insects, the grain beetle Tenebrio molitor). Weak physical and chemical factors were used: electromagnetic radiation, constant, alternating, and combined magnetic fields, and low concentrations of solutions of neuropeptides. It is shown that these influences are characterized by instability and opposite directions (from stimulatory to inhibitory). The dependence of the effects on external factors and the course of internal processes was established.     

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

Immunocytochemical identification of serotoninergic neurons in planaria <Emphasis Type="Italic">Girardia tigrina</Emphasis>

Flatworms occupy an important position among simple organisms, which were first in the evolution having bilateral symmetry and centralized nervous system. This paper provides evidence of the presence of a biogenic amine serotonin in free-living flatworms planarians Girardia tigrina (Turbellaria, Platyhelminthes). Using immunohistochemical method, fluorescence and confocal laser scanning microscopy, we have identified serotonin neurons and their fibers using planarian whole-mount preparations and got important information about distribution of serotoninergic components in their body. Information on the number and size of serotonin-immunopositive neurons in the brain ganglion of G. tigrina and on the distribution density of serotoninergic neurons in the central nervous system of worms is presented for the first time. The published data concerning the serotoninergic signalization in flatworms are briefly overviewed.     

Studies on the Effect of a Weak Electromagnetic Field on Pharynx Regeneration in Dugesia tigrina Planarians

We have studied the effect of a weak electromagnetic field on the morphogenesis of the planarian Dugesia tigrina. The regeneration of the pharynx was examined after its amputation. We have determined the rate of the appearance of the food response of a new pharynx. Experimental conditions were varied, such as dose and duration of irradiation, season, and time of irradiation after surgery. The results of experiments conducted with 2966 planarians have shown that weak electromagnetic field has various effects, which appear either as stimulation of regeneration or as its inhibition. In some experiments, there was no effect at all. These differences depend on numerous factors and may be modulated.     

Regeneration processes in various species of planarians

Blastema growth and functional maturation of the pharynx during regeneration in various planarian species were compared. The intensity of blastema growth was highest in Polycelis tenuis; the lowest, in Schmidtea mediterranea. In the sexual and asexual races of Girardia tigrina blastema growth differed inconsiderably. The function of the pharynx during the regeneration of caudal fragments lacking pharynx was manifested in G. tigrina in the usual amount of time, while in the regeneration of head fragments lacking pharynx, this function occured earlier. In other planarian species of the other two typed, the times of pharynx regeneration had no regular character and took longer compared to the same process in G. tigrina.     

Effect of weak and ultraweak combined magnetic fields and low-intensity microwaves on regeneration in the planarian <Emphasis Type="Italic">Dugesia tigrina</Emphasis>

Regeneration (blastema growth) in Dugesia tigrina was accelerated if prior to transection the planarians were exposed to a weak constant magnetic field (42 μT) combined with an ultraweak alternating magnetic field (40 nT, 3.7 Hz); lesser stimulation was obtained with weak microwaves (100 μW/cm² at 36 GHz). Field exposure after transection produced only half of the effect (magnetic field) or none at all (microwaves).     

cDNA cloning and partial sequencing of homeobox genes in Dugesia japonica

In the freshwater planarian Dugesia japonica, four types of cDNAs of homeobox-containing genes have been isolated by screening a cDNA library using a homeobox guessmer. Partial sequencing analysis of two types of cDNAs revealed that one was a homolog of Dth2 which is a homeobox gene in Dugesia tigrina and another was similar to Distal-less gene in Drosophila. This suggests that planarians have many homeobox genes.     

Regeneration in two freshwater planarian species exposed to methylmercury compounds

The regeneration rate was studied by the morphometric method in planarians Polycelis tenuis and Dugesia lugubris after different periods of exposure on food substrates with high (0.30–0.50 mg/kg wet weight) and low (0.02–0.07 mg/kg) concentration of methylmercury compounds. The planarian growth and the ratio of regenerating tissue area to the total fragment area after transverse cutting were evaluated. The rate of size increment was lower in animals with high level than with low level of methylmercury than with low level of methylmercury. The highest relative and absolute body area increment after amputation was observed in D. lugubris and P. tenius, respectively. Thus, natural methylmercury compounds were shown to inhibit tissue regeneration in planarians in a dose-dependent manner.     

Asexual reproduction,regeneration, and somatic embryogenesis in the planarian Dugesia tigrina (Turbellaria)

In reviewing recent research published in Russian on regeneration and asexual reproduction, the following morphogenetic processes in the planarian Dugesia tigrina are considered: 1) regeneration of lost parts of the body; 2) regeneration of the whole worm from fragments of the body, either by normal regeneration when the inital polarity of the fragment is retained or by somatic embryogenesis when one or more new axes of polarity arise; 3) somatic embryogenesis, or development of individuals from somatic cells; 4) hypermorphosis, or the presence of more than the usual number of organs or body parts, a process that can be interpreted in terms of somatic embryogenesis; and 5) asexual reproduction. Some morphological, biochemical, and physiological studies of the division zone in D. tigrina demonstrate peculiarities of a local breakdown of integrative functions, a breakdown which in turn causes division of the individual to take place at this zone; timing of division is controlled by the organism as an integrated whole.