A fine structural study of regeneration after fission in the planarian Dugesia japonica

We examined morphologically the process of regeneration before and after fission in a sexual strain of the freshwater planarian Dugesia japonica. Usually fission takes place in the post-pharyngeal region. Decapitation significantly accelerates the rate of fissioning. When decapitated worms were treated with substance P and neuropeptide K separately, the rate of fission markedly decreased in both cases. Before the onset of fission, a presumptive region of fission was recognized in the post-pharyngeal portion where undifferentiated cells, regenerative cells and newly differentiated cells were localized. Moreover a functional network of fixed parenchyma cells was noted in this region. After fission, cell distribution in the blastema became quite different from that of artificially amputated worms. This difference seems to be due to the process that occurs in the presumptive region of fission. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of planarian regeneration by melatonin

Melatonin, which is a substance produced by the pineal body in vertebrates, inhibited regeneration in the planarian Dugesia japonica japonica Ichikawa et Kawakatsu. When decapitated planarians were maintained in a 1 mmol dm^–3 solution of melatonin, formation of the head was retarded; formation of the eyes, however, was not disturbed. Similarly in animals from which the tail was cut, regeneration of the tail was retarded if the animals were kept in melatonin solution of 1 mmol dm^–3. The effect was reversible once the melatonin was removed. Retardation of regeneration did not occur with similar application of three melatonin derivatives, serotonin hydrochloride, N-acetylserotonin, and 6-hydroxymelatonin. Melatonin endogenous to the planarian could be demonstrated by means of radio-immunoassay and was more abundant in the head region than other regions of the body. Melatonin, thus, appears to play a role in regulating regeneration in planarians and conceivably provides positional information in that process.     

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.     

Regulation of melatonin production by light,darkness, and temperature in the trout pineal

Summary The pineal gland of the rainbow trout, Salmo gairdneri, when kept under in vitro perifusion culture conditions, displays a consistently elevated level of melatonin production in darkness (Gern and Greenhouse 1988). Upon light exposure melatonin production falls and stabilizes at a new lower level that is dependent upon the irradiance of the stimulus. To achieve the maximal response for each irradiance, the duration of the stimulus must exceed 30 min. The response amplitude is maximally sensitive to photons presented over durations of 30–45 min; is very insensitive to shorter light exposures; and is maintained with no evidence of adaptation over longer exposures. Temperature plays a role in regulation of melatonin production both in darkness and during light exposure; increased temperature increases melatonin production in darkness and also increases the sensitivity of the response to light. The action spectrum for the response is best fit by the Dartnall nomogram for a vitamin A₁ based rhodopsin with peak sensitivity near 500 nm. The possible adaptive significance of control of melatonin synthesis by light and temperature is considered.Abbreviations LD lightdark cycle - RIA radioimmunoassay - I ¹²⁵ Iodine - HIOMT hydroxyindole-O-methyltransferase     

Effects of exogenous melatonin on the reproductive activities of Nile tilapia,Oreochromis niloticus

Gonadal development and sexual maturation of fish are often related to photic conditions. We attempted to utilise the direct application of melatonin to elicit similar effects in the absence of photoperiod manipulation. We found no significant differences in somatic growth between melatonin-treated groups and controls, indicating that melatonin administration did not negatively affect the growth of the experimental fish. Treatment with low-dose melatonin (0.3 mg/kg BW) resulted in a decrease in spawning frequency, number of spawned eggs and gonadosomatic index in female tilapia. In male tilapia, low-dose melatonin was associated with a decrease in sperm count, spermatocrit and spermatozoa activity index, implying that exogenous melatonin might mimic the effects of shortened light photoperiod, which suppresses reproductive activity in this species. These results suggest the possibility that an optimised dose of melatonin treatment could be used to control the spawning behaviour of this species.     

A Wnt11 and Dishevelled signaling pathway acts prior to injury to control wound polarization for the onset of planarian regeneration

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Insights into the histology of planarian flatworm Phagocata gracilis based on location specific,intact lipid information provided by GCIB-ToF-SIMS imaging

Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight – Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.     

Proportion regulation in the planarian,Dugesia tigrina following regeneration of structures

Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Day-night behavior in river entry of kutum and its relation to melatonin

Reproduction in most fish species is a scheduled phenomenon and spawning occurs at a precise time ensuring maximum survival of offspring. During the spawning season, reproductive activities of fish species and their daily entry to rivers are influenced by environment. Number of Rutilus frisii kutum entered a river discharging to Caspian Sea was analyzed to investigate the association between timing of river entry and day–night changes in environmental variables. Four thousand five hundred and sixty seven ascending adult kutum were collected in Khoshkrood River between March and May 2012. Physicochemical parameters were recorded and Melatonin levels were measured in a hundred migratory kutum over day and night during the spawning period. A significant effect of daytime on river entry was observed, as more fish entered the river at night. Melatonin levels decreased to the minimum level during peak days of migration, suggesting variations in melatonin levels can help to harmonize the daily rhythm in kutum migration.     

Temperature, light intensity and plasma melatonin levels in juvenile Atlantic salmon

Plasma melatonin synthesis in juvenile Atlantic salmon Salmo salar decreased with increasing light intensity. Melatonin profiles reflected accurately the photoperiod under which the salmon were maintained. Groups maintained at 12°C showed significantly higher ( P < 0·01) levels of dark phase plasma melatonin compared with the groups maintained at 4°C.     

Structure and function of the reticular cell in the planarian Dugesia dorotocephala

Structural and functional characteristics of the reticular cell in the planarian Dugesia dorotocephala were studied by light and electron microscopy. Since the reticular cells have numerous glycogen granules, lipid droplets and some lysosomes in their cytoplasm, they are easily distinguishable from other cell types. They migrate into the injured tissue, cover the injured mesenchyme, and also phagocytize debris of degenerating cells. The reticular cells also recognize foreign invaders such as bacteria. The larger aggregates of killed bacteria are encapsulated by reticular cells and eliminated into the intestine, whereas small aggregates are phagocytized by reticular cells. When cell wall extract of bacteria was inserted into the planarian body before insertion of killed bacteria, reticular cells were found to respond more quickly and vigorously to subsequent insertion of killed bacteria, indicating that the reticular cell has an immune response memory. When planarians were treated with 0.3 ppm cadmium sulfate and 0.01 ppm TPA, reticuloma tumors were induced in 76% of exposed planarians, indicating the similarity to blood cell diseases in mammals such as leukemia or lymphoma which are also induced by TPA. When these tumors were transplanted into normal hosts, the tumor cells were attacked by host reticular cells. These observations indicate that planarian reticular cells are primitive blood cells, playing important roles in nutrient transportation, homeostatic control of cells, and in defence and immune surveillance systems.     

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.     

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 µg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 µg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.     

Circadian rhythms of corneal mitotic rate,retinal melatonin and immunoreactive visual pigments,and the effects of melatonin on the rhythms in the Japanese quail

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 1212), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 1212. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 1212, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 1212. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 1212, but did not induce a rhythm under LL. The ratio of photoreceptor cells with positive staining to anti-visual pigment antibodies at the Golgi region was not affected by melatonin injection.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - DD constant darkness - Io-mAb monoclonal antibodies against chicken iodospin - LD light-dark - LL constant light - mRNA messenger ribonucleic acid - PBS phosphate buffer solution - Rh-As antiserum against bovine rhodopsin - SCN suprachiasmatic nucleus - T transducin - T transducin      

Weak extremely-low-frequency magnetic fields and regeneration in the planarian Dugesia tigrina

Extremely-low-frequency (ELF), low-intensity magnetic fields have been shown to influence cell signaling processes in a variety of systems, both in vivo and in vitro. Similar effects have been demonstrated for nervous system development and neurite outgrowth. We report that regeneration in planaria, which incorporates many of these processes, is also affected by ELF magnetic fields. The rate of cephalic regeneration, reflected by the mean regeneration time (MRT), for planaria populations regenerating under continuous exposure to combined DC (78.4 μT) and AC (60.0 Hz at 10.0 μT _peak) magnetic fields applied in parallel was found to be significantly delayed (P ? 0.001) by 48 ± 1 h relative to two different types of control populations (MRT ? 140 ± 12 h). One control population was exposed to only the AC component of this field combination, while the other experienced only the ambient geomagnetic field. All measurements were conducted in a low-gradient, low-noise magnetics laboratory under well-maintained temperature conditions. This delay in regeneration was shown to be dependent on the planaria having a fixed orientation with respect to the magnetic field vectors. Results also indicate that this orientation-dependent transduction process does not result from Faraday induction but is consistent with a Ca²⁺ cyclotron resonance mechanism. Data interpretation also permits the tentative conclusion that the effect results from an inhibition of events at an early stage in the regeneration process before the onset of proliferation and differentiation. © 1995 Wiley-Liss, Inc.     

Protein phosphorylation and the role of Ca2+ in planarian turbellarian regeneration

During planarian regeneration, relationships in timing were established between variations of calcium and calmodulin contents on one hand, and protein phosphorylation and stimulation of DNA and RNA synthesis on the other. Special attention was paid to changes in histone phosphorylation in regenerating fragments. Using in vitro experiments on dissociated planarian cells, we demonstrated causal relationships between these events. In particular, the key role of Ca²⁺ in the activation of protein kinases and in the initiation of DNA synthesis was emphasized.     

Production of cell- and tissue-specific monoclonal antibodies in the freshwater planarian Phagocata vivida

To provide a tool for studying regeneration in planarians, we have produced monoclonal antibodies against a variety of cells and tissues of the freshwater planarian Phagocata vivida (Ijima et Kaburaki). We obtained five kinds of monoclonal antibodies specific, respectively, to 1) the excretory system, 2) nerve cells, 3) rhabdoid-forming cells and body-surface mucus, 4) gastrodermal and epidermal cells, and 5) male germ cells and epidermis.     

Weak extremely-low-frequency magnetic field-induced regeneration anomalies in the planarian Dugesia tigrina

We recently reported that cephalic regeneration in the planarian Dugesia tigrina was significantly delayed in populations exposed continuously to combined parallel DC and AC magnetic fields. This effect was consistent with hypotheses suggesting an underlying resonance phenomenon. We report here, in a parallel series of investigations on the same model system, that the incidence of regeneration anomalies presenting as tumor-like protuberances also increases significantly (P < .001) in association with exposure to weak 60 Hz magnetic fields, with peak intensities ranging between 1.0 and 80.0 μT. These anomalies often culminate in the complete disaggregation of the organism. Similar to regeneration rate effects, the incidence of regeneration anomalies is specifically dependent upon the planaria possessing a fixed orientation with respect to the applied magnetic field vectors. However, unlike the regeneration rate effects, the AC magnetic field alone, in the absence of any measurable DC field, is capable of producing these anomalies. Moreover, the incidence of regeneration anomalies follows a clear dose-response relationship as a function of AC magnetic field intensity, with the threshold for induced electric field intensity estimated at 5 μV/m. The addition of either 51.1 or 78.4 μT DC magnetic fields, applied in parallel combination with the AC field, enhances the appearance of anomalies relative to the 60 Hz AC field alone, but only at certain AC field intensities. Thus, whereas our previous study of regeneration rate effects appeared to involve exclusively resonance interactions, the regeneration anomalies reported here appear to result primarily from Faraday induction coupling. These results together with those reported previously point to two distinct physiological effects produced in regenerating planaria by exposure to weak extremely-low-frequency (ELF) magnetic fields. They further suggest that the planarian, which has recently been identified elsewhere as an excellent system for use in teratogenic investigations involving chemical teratogens, might be used similarly in teratogenic investigations involving ELF magnetic fields. © 1996 Wiley-Liss, Inc.