Relationship between variations of cAMP,neuromediators and the stimulation of nucleic acid synthesis during planarian (Polycelis tenuis) regeneration

Regenerating planarians of the species Polycelis tenuis have been studied with respect to the two distinct phases which occur during regeneration, viz., the first 24 h involving cellular activation, and the following days when differentiation of the blastema occurs after a period of cellular proliferation. We have studied particularly the biochemical events that control regeneration with respect to the nature of the signals which induce cellular activation, the membrane receptors of these signals, and the consequences of these signals for the cellular metabolism of DNA, RNA and proteins. The roles of neurohormones such as serotonin, dopamine, and noradrenaline are assessed, and a provisional model of the process of cellular activation is proposed which takes account of all the information that is now available concerning planarian regeneration.     

GABA in the nervous system of the planarian Polycelis nigra

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates and it has a similar inhibitory role in several invertebrate taxa. The transmitters serotonin, octopamine, catecholamines and histamine are present in flatworms while evidence for GABA is still lacking. Therefore, we have studied the occurrence of GABA-like immunoreactivity (IR) in the planarian nervous system. Specimens of Polycelis nigra were fixed in 4% 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide with 2% paraformaldehyde. The GABA-antiserum was raised in rabbits against GABA conjugated to keyhole limpet hemocyanin. Preabsorption with GABA-ovalbumin conjugate abolished all IR. The results were further confirmed with an monoclonal antibody and high pressure liquid chromatography (HPLC). In P. nigra GABA-like IR was seen as long, often varicose, sparsely distributed fibers in the ventral longitudinal nerve cords. IR was also located in a few cell somata in the brain and in the neuropil of the brain. The IR was restricted to the central nervous system and was absent in peripheral nerves and plexuses. The HPLC analysis supported the presence of GABA.Our results suggest that GABA is an interneuronal transmitter in P. nigra. The results also suggest a phylogenetically old origin of GABAergic neurotransmission.     

Use of the p-nitrophenyl phosphate method for the demonstration of acid phosphatase during starvation and cell autolysis in the planarian Polycelis tenuis Iijima.

Synopsis Acid phosphatase activity is demonstrated employingp-nitrophenyl phosphate as substrate and lead acetate as coupler. The fine structural localization of the enzyme in starved planarian tissues is described. The method is used to pin-point starvation-induced acid phosphatase activity in relation to autophagy and crinophagy in the gland cells; autophagy, autolysis and cell death in parenchymal and gastrodermal cells and basement membrane lysis. Attention is also payed to the demonstration of muscle lysis. The histochemical implications of the method are discussed.     

Further survey of chromosomal polymorphisms in the freshwater planarian Polycelis auriculata

Although the chromosomal number of Polycelis (Seidlia) auriculata Ijima et Kaburaki is basically 2n = 6, this species shows remarkable chromosomal polymorphisms in different populations. Among worms collected at 30 stations in the central part of Japan's Main Island and in Hokkaido, we found five new karyotypes, considered to be variations of tetraploidy and diploidy: 4n?fis¹. + 1 = 3SM + 2A + 3T₁ + 2T₂ + 4M = 14, 4n?fis².(B) = 2SM + 2A + 4T₁ + 2T₂ + 4M + (0?2B) = 14 –16, 4n?fis². + 1 = 2SM + 2A + 4T₁ + 2T₂+5M= 15, 4n?fis².?2 = 2SM + 2A + 4T₁ + 4M = 12, and 2nM- = 2SM + 2A = 4 (where SM = submetacentric; A = acrocentric; T₁ and T₂ = telocentric; M = metacentric; B = B-chromosome; fis¹. and fis². = occurrence of one or two centric fissions, respectively; and M- = deletion of a metacentric chromosome). These karyotypes exist singly in one individual or as various mixoploids within the same individual. No individual having only karyotype 2nM- has yet been found. This brings the total number of known karyotypes in P. auriculata to 17; these can be arranged in an order that we believe reflects their evolution.     

Specific features of eye regeneration in multiocular planarians Polycelis tenuis

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.     

Cell Death and Acid Phosphatase Activity in the Regenerating Planarian Polycelis tenuis Iijima

A combination of microscopical, cytochemical, and biochemical techniques have been employed to study the changes occurring during the first seven days of blastema formation and regeneration after decapitation in adult Polycelis tenuis worms. Fine structural data reveal evidence of cell fragmentation, selective cell deletion, and phagocytosis at and below the wound surface. Initially, (0–12 h regeneration) cell debris is phagocytosed by intact parenchymal and gastrodermal cells near the cut surface which is later sealed (24 h) by a stretching of marginal epidermal cells. Wound sealing is followed by a migration of newly differentiated rhabdite cells into the epithelium. Morphological evidence of a selective cell autolysis precedes evidence of an accumuluation of lipid and glycogen reserves in the parenchymal and gastrodermal cells and the later (48 h regeneration time) aggregation of undifferentiated mitotically active neoblasts beneath the wound.
Biochemical data reveal an early period of high acid phosphatase (p-nitrophenyl phosphatase and sodium-β-glycerophosphatase) activity 3–12 h after injury, followed by a further intense period of activity at 44–48 h after decapitation. The coincident cytochemical data show an increased level of acid phosphatase activity associated with cell lysis and death in the wound and blastema zone and also with the digestion of phagocytosed cell debris.     

The geographical distribution of Polycelis (Polycelis) sapporo and Seidlia schmidti in the Far East

Polycelis (Polycelis) sapporo (Ijima et Kaburaki, 1916) is a non-stenothermal species, common in Hokkaidô (including all of the adjacent islands), Northern Japan, except in the alpine region. It is also distributed in Aomori Prefecture in the northernmost part of Honshû. To the south, its range is bounded by a line from the base of the Tsugaru Peninsula, over Mt. Iwaki, Mt. Shirakami, Mt. Tashiro, Lake Towada, Mt. Hakkôda, the Natsudomari Peninsula, to the west of Mutsu and Ôhata in the Shimokita Peninsula. In Russia, the species is recorded only from the southern part of Sakhalin. Seidlia schmidti (Zabusov, 1916) is a stenothermal species found in cold-water biotops. It occurs in the North and East of Hokkaidô including the Shiretoko Peninsula and Rishiri Island. In Russia, the species is recorded from the southern part of Sakhalin, Primorskiy, Habarovsk including the base of the Chukotskiy Peninsula, and the Kamchatka Peninsula (and Bering Island).     

The adenylate cyclase system of planaria Polycelis tenuis: activation by serotonin and guanine nucleotides

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5'-(beta-gamma-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a KA of 2 micrometer and a Hill coefficient of 0.4. In the presence of 10 micrometer Gpp(NH)p, these values became 45 nM and 1.5, respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 micrometer in the absence of Gpp(NH)p and at 5.0 micrometer in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin is normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylate cyclase activity plays a physiological role in this species.     

Studies on the speciation of the European freshwater planarians Polycelis nigra and Polycelis tenuis based on the analysis of enzyme variation by means of iso-electric focusing

Polycelis nigra Ehrenberg and Polycelis tenuis Ijima differ morphologically and karyologically. No difference, however, was found in the isozyme pattern of malate dehydrogenase and tetrazolium oxidase, indicating a close relationship. Most sibling species differ at half of the loci.It could be deduced that the reproductive behaviour of a single population of Polycelis nigra in a Dutch pond was not panmictic. Two genetically different strains retained their identities during two years of observation. If pseudogamy occurs in this diploid planarian, the presence of heterozygous specimens indicates the absence of a true meiosis.The iso-electrofocusing technique by which these population-genetical studies were carried out, also lends itself to a comparison of overall protein banding patterns. The membrane proteins especially are conservative. The sodium dodecyl sulphate extracted proteins of Polycelis nigra-tenuis, Planaria torva and Phagocata vitta were very similar, while their water soluble proteins were not. This technique may be of great help in taxonomic studies of the higher taxa.     

Karyological analysis of two allopatric populations of planarian Polycelis felina (Daly.) in Croatia

The results of preliminary karyological investigations of two geographically distant and morphologically slightly different Polycelis felina (Daly.) populations from central Croatia are reported. The results have shown that individuals of both populations are diploids, with the same chromosome number in their neoblasts (2n=18). Their karyotypes were composed of nine chromosome pairs, three of them are metacentric and the other six are submetacentric. Statistical evaluation of data indicated that both populations of Polycelis felina (Daly.), despite minor differences observed between them, belong to the same karyological biotype.     

The specific cytochemical demonstration in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups

Summary A technique is described for the localization in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups. In this technique the sugar residues are oxidized by periodic acid and the resulting aldehydes condensed with pentafluorophenylhydrazine under specified conditions. Further increase in specific electron density is achieved by treating the hydrazone end-product with ammonium sulphide followed by osmium tetroxide to form an osmium black.The technique has been applied to liver and small intestine cells in which glycogen, sialomucins and sulphated mucosubstances reacted especially strongly. A marked positive reaction has also been obtained from the interstitial cell matrix and from the Golgi apparatus and multivesicular bodies of the intestinal epithelial cells.The reaction can be prevented by the omission of the periodate oxidation and, if due to glycogen, by prior treatment with diastase.     

Nuclear distribution in Alternaria tenuis

Nuclear distribution and behaviour during vegetative growth and spore formation inAlternaria tenuis was studied utilising the HCl — Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.     

Allopatric and sympatric populations of Polycelis nigra in competition with P. tenuis (Turbellaria; Tricladida)

Two allopatric populations of Polycelis nigra (MuUller), one from lowland productive Llyn Maelog on Anglesey and the other from upland unproductive Llyn Dinas in Snowdonia, were each subjected to competition in the laboratory from the same Polycelis tenuis Ijima population from productive Llyn Llygeirian, also on Anglesey, where it coexists with P. nigra. The triclads were fed weekly on crushed Asellus (Isopoda) and their biomasses calculated monthly over five months. Results suggest that sympatric P. nigra are more successful competitors against P. tenuis than allopatric P. nigra. The data also suggest that the inferior competitive ability of allopatric P. nigra may be a consequence of both exploitative and interference competition. Coexistence of P. nigra with P. tenuis appears to increase P. nigra's tolerance to environmental stress; the current experiments and observations during several decades of research have indicated poorer survival and growth in allopatric than sympatric populations.     

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.