Triturus Cristatus Carnifex Research Articles

Leptin, leptin receptors and ACTH immunoreactivities are present in the gastrointestinal tract and the neural tube of tadpoles of the newt Triturus.

Leptin, its receptor and ACTH were detected by immunohistochemistry in the gastrointestinal tract and the neural tube of the amphibian urodele, Triturus cristatus carnifex, during development. These molecules were found after hatching of tadpoles, starting from stage 41. In the gastrointestinal tract, cells immunoreactive to leptin and its receptor were first revealed in the stomach, the liver and the gut and then in the pancreas. Both immunoreactives were colocalized in the same cells in some areas. Immunostaining for ACTH appeared at stages 43/45 in the stomach, the gut and the pancreas. In adjacent sections, a few cells immunoreactive to both ACTH and leptin receptor were detected. A few cells were immunoreactive to both insulin and leptin receptor. Immunoreactivities to leptin and its receptor were also found in adjacent sections of the neural tube, often colocalized in the same cell. Moreover, in prosencephalon, mesencephalon, rhomboencephalon and spinal cord, ACTH-immunoreactive cells were detected in the same areas as the leptin receptor immunoreactive cells. These results suggest the existence of a neuroendocrine network in newt tadpoles both at the central level, where it resembles that of mammals, and at the peripheral level, where it may act locally to regulate food intake and metabolism, e.g. yolk digestion.

Variability of DNA methylation pattern in somatic and germ cells in male newt (Amphibia, Urodela) Triturus cristatus carnifex

In a survey of several mammalian genomes, namely humans, rodents and bovines, the differences in the 5-methylcytosine (m 5C) content show that repeated DNA sequences from sperm were undermethylated and from various somatic tissues were heavily methylated. This report shows a pattern of methylation in male newt (Amphibia, Urodela) Triturus cristatus carnifex ( T. c. c.) unlike that so far described by other authors in mammals. Using methylation sensitive and insensitive enzymes ( HpaII and MspI) and successive 3′ terminal labelling (fill-in), we found a greater degree of DNA methylation in premeiotic germ and sperm cells compared to somatic tissue such as hepatocytes. Furthermore the degree of total DNA methylation in spermatozoa appears somewhere between premeiotic germ cells and somatic tissue. Blot hybridization shows that two highly conserved repetitive sequences in amphibian T. c. c., pTvm1 and pTvm8, contribute significantly to the degree of DNA methylation, suggesting a function for these sequences, such as a role in transcriptional regulation.

Lampbrush chromosomes and chiasmata of sex-reversed crested newts.

Triturus cristatus carnifex provides a particularly clear example of sexual dimorphism for chiasma frequency and localisation. Oocytes from normal XX females routinely carry one proximal chiasma on each arm of their lampbrush bivalents. Spermatocytes from normal XY males have more numerous and relatively distal chiasmata. Lampbrush chromosomes from the oocytes of sex-reversed XY neofemales are found to resemble those from normal oocytes in having one proximal chiasma on each bivalent arm. A comparison of particular markers on the heteromorphic long arm of chromosome 1 provides evidence to equate the lampbrush 1A to somatic 1A, and confirms previous reports that lampbrush chromosome 1A is slightly longer than 1B. The XY sex bivalent of neofemales does not show any obvious heteromorphy of recognised marker loops.

Data on haemodynamics of splenic circulation in the newt

In the newt, Triturus cristatus carnifex (Laurenti), the spleen normally contains a reserve of erythrocytes which varies according to the respiratory condition of the animal. In chlorobutanolanaesthetized specimens, histological analyses and in vivo observations following the injection into the bloodstream of fluorescent microspheres 10 μm in diameter (FluoresbriteTM YG, Polysciences Inc., USA) show that, in steady conditions, the stored red blood cells are not segregated in the spleen but pass through the organ with a very slow flow; their time of transit in a spleen maintained in a state of maximum congestion is 41.5 ± 14.2 minutes (mean ± standard deviation.

Consequences of splenectomy on the compensatory mechanism of cutaneous respiration in the newt

AbstractThe spleen of the newt Triturus cristatus carnifex (Laurenti) hoards erythrocytes when the animal is well oxygenated and releases these into circulation when the animal becomes hypoxic. This mechanism allows the newts, amphibians with prevalently cutaneous respiration, to adapt to both environmental and metabolic changes.Splenectomized specimens were anesthetized with chlorbutol (to eliminate movement and lung respiration) and observed in five different conditions: at 18°C in humid air, stirred water, and still water, and at 6°C and 33°C in humid air. The study demonstrates that the newt does not have substitute sites for erythrocyte accumulation. However, though the spleenless specimens are incapable of controlling their cutaneous oxygen supply, they can regulate their blood volume, which diminishes with decreasing temperature causing marked variations in the erythrocyte concentration and hematocrit value.The splenectomized specimens, raised for over a year at 18°C, present typically steatosic livers, with lipid droplets and extremely low hepatocyte glycogen levels, indicating a state of chronic hypoxia due to the prevalently aquatic habits of the newt. In fact, the spleenless newts have significantly fewer erythrocytes (P < 0.001) than the unoperated animals‐‐a total volume of 2.27 ± 0.46 ml per 100 g of body weight compared to 2.74 ± 0.29 (mean ± standard deviation). The limited quantity of erythrocytes can provide adequate oxygen only at low temperatures and in an airy environment or in moving water, while an immobile animal in still water undergoes hypoxia due to the formation around it of an hypoxic =diffusion boundary layer= caused by the low coefficient of oxygen diffusion in water. © 1993 Wiley‐Liss, Inc.

Splenic respiratory compensation and blood volume in the newt

In the newt Triturus cristatus carnifex, reversible increase in size of the spleen is linked to the respiratory state of the animal: when the newt is exposed to the air, and thus well oxygenated, the spleen hoards erythrocytes; when immersed in still water, in an hypoxic state, the spleen releases erythrocytes into the bloodstream. In chlorobutanol‐anaesthetized specimens exposed to the air, the maximum size reached by the spleen diminishes with a rise in temperature up to the disappearance of all congestion at 33°C. The blood volume of newts kept in humid air at 6°C and 18°C after anaesthesia varies from about 6–9 ml per 100 g of body weight, while the red blood cell count and haematocrit value remain stable. In anaesthetized specimens kept in still water at the same temperatures the blood volume is stable, at about 7 ml per 100 g, but the red cell count and haematocrit are notably higher. At 33°C, a critical temperature for the newts, the specimens in still water succumb while those in air present the same blood volume as at 18°C, but have a higher erythrocyte count and haematocrit value.

Hematological changes in the newt during the process of splenic decongestion in reply to hypoxia

Abstract Specimens of the newt, Triturus cristatus carnifex (Laurenti), anesthetized with chlorobutanol and exposed to humid air at 6° C for 300 min, accumulate about 50 of their red blood cells (RBC) in the spleen. When the newts are transferred to still water at 18° C, the spleen releases the RBC back into circulation, emptying itself out completely. This process counteracts the hypoxia caused by both the increase in temperature and low coefficient of oxygen diffusion in water. The analysis of the RBC counts, hematocrit value and percentage volumes of the spleen, blood and plasma (the last measured colorimetrically after injecting 0.05 ml of Evans blue in the conus arteriosus) in groups of newts sampled at various periods during the spleen decongestion reveals the trend of the compensatory process. The emptying out of the spleen takes less than an hour, causing significant increases in the RBC concentration after 15 and 30 min of immersion; the counts then undergo an inflexion which becomes significant ...

Immunocytochemical identification of growth hormone- and prolactin-producing cells in the hypophysis of the newt Triturus cristatus carnifex Laur

Single indirect immunocytochemical methods (immunofluorescence, PAP, and ABC) and double sequential staining (ABC followed by immunofluorescence) were used to localize GH- and PRL-producing cells in the pituitary distal lobe from Triturus cristatus. The following antisera were employed: rabbit anti-ovine PRL, anti- Rana catesbeiana PRL, anti-ovine GH, anti-bovine GH, and monkey anti-rat GH. A cell population corresponding to type-2 acidophils localized in the dorsal and central region, under the intermediate lobe, immunoreacted with GH antisera. Both ovine and bullfrog PRL antisera labeled a large cell population, corresponding to type-1 acidophils, predominantly localized in the ventral anterior two-thirds of the gland. The pattern of localization shown by the two cell types, although consistent with the majority of data on adult amphibians, disproves the findings obtained on the same species by other authors.

Brain-capillary architecture of the newt, Triturus cristatus carnifex (Caudata: Salamandridae): Scanning electron-microscopical study of vascular corrosion casts.

The brain-microvascular architecture of the newt (Triturus cristatus carnifex) was studied by scanning electron-microscopical examination of microvascular corrosion casts. It is characterized at the leptomeningeal level by a superficial network of single vessels that leave the meningeal sheath to form hairpin-shaped capillary loops. These loops penetrate the nervous parenchyma to varying depths and are variously bent, inclined, and twisted. Each hairpin-like loop consists of descending and ascending limbs connected by a distal U-shaped segment. The limbs are of equal diameter and lack physical differentiation. Some loops branch in a secondary loop, but there are no intraparenchymal connections with analogous surrounding structures. On vascular casts we observed detailed imprints of endothelial cell nuclei. This microvascular architecture is present in the whole brain except in leptomeninx and choroidal plexuses. The vascular pattern of T. cristatus carnifex resembles that of apodans. However, it differs from the anuran condition, which is characterized by a three-dimensional parenchymal network and from that of Ambystoma mexicanum which displays an intermediate condition between a single-capillary network and a capillary-loop model. At the level of paired capillaries, a counter-current exchange system may occur between the two limbs of the same loop, an arrangement that could promote uniformity of extracellular fluid composition in parenchyma.

Effect of temperature on the splenic mechanism of respiratory compensation in the newt

AbstractThe spleen of the newt Triturus cristatus carnifex (Laurenti), an urodele amphibian, hoards erythrocytes when the animal is well‐oxygenated and releases them into the bloodstream when the animal is in a hypoxic condition. Between the extremes of congestion and decongestion the weight of the spleen varies up to a ratio of 6:1. However, the degree of the phenomenon depends on the temperature, which, in ectotherms, regulates metabolic rate and thus oxygen demand. In order to assay the degree of hypertrophy reached by congested spleens in standard respiratory conditions, groups of eight animals—previously anesthetized by immersion for 20 minutes in a 0.2% solution of chlorbutol in tap water at 18°C—were exposed to 100% humid air in a thermostatic chamber set at a temperature of 6, 12, 18, 24, 27, 30, or 33°C. Each animal remained at the set temperature until surpassing the minimum time necessary for maximum hypertrophy to be reached, equal to the time necessary for 4,000 heartbeats. At 33°C the spleens are almost completely bloodless, while at the other temperatures their hypertrophy from congestion increases linearly as the temperature diminishes, reaching maximum values at 6°C. Though the progressive congestion of the spleen as the temperature descends is due mainly to blood cells and only very slightly to plasma, erythrocyte concentration in the blood remains constant at all the lower temperatures up to that of anesthesia (18°C); this phenomenon implies variability of blood volume in the newt. Instead, above 18°C, the values of the hematic parameters increase as the temperature rises, reaching a maximum increment of about 30% by 27°C.

Effect of spleen congestion and decongestion on newt blood

Experiments on the newt Triturus cristatus carnifex have shown that the spleen of specimens, either anaesthetized with chlorobutanol or in a normal condition, stores red blood cells when the animal is well oxygenated and releases these into circulation under conditions of hypoxia. The extreme limits of congestion and decongestion, commonly encountered in nature, can be obtained in the laboratory by exposing the animals to the air or by immersing them in still water. During the shift from a congested to decongested state, the spleen is reduced to less than one‐fifth of its weight, while the erythrocyte concentration and correlated parameters in the blood stream increase by more than 60%. Splenic regulation of the circulating erythrocyte concentration compensates not only for variations in oxygen supply linked to environment, it may also compensate for the cyclical variations in red cell production of this amphibian, whose erythropoietic tissue activity is intermittent and subject to seasonal variations.

Relationships between spleen and respiration in the newt.

Specimens of newt, Triturus cristatus carnifex (Laurenti), anesthetized by submersion in 0.2% chlorbutol in tap water for 15 min, and then placed out of water in a damp terrarium, show hypertrophy of the spleen that in 2 hr gradually increases from 0.31 +/- 0.12%with respect to body weight to 1.56 +/- 0.26% (means and standard deviation calculated for groups of six animals). Other anesthetics either do not produce hypnosis (Veronal), do not have a prolonged enough effect (ethyl ether, chloroform), or induce vasodilatation, which prevents hypertrophy (MS-222, urethane). The spleen hypertrophy, seen histologically to be due exclusively to blood congestion, is not caused by either a pharmacological effect of the chlorbutol or by the hypnotic state, as it does not appear in submerged anesthetized animals, unless the water is constantly stirred by a magnetic agitator, and can be reversed depending on the ventilation of the animal's skin. The spleen hoards blood when oxygenation is good (in air or stirred water) and releases this supply in the bloodstream when oxygenation is insufficient (in still water). The hypoxic "diffusion boundary layer," which, in still water, forms around the immobile newts, hampers respiratory exchange and stimulates the spleen contraction. This mechanism and its relationship to oxygenation has been demonstrated statistically in unanesthetized newts as well, in both air and water, despite the interference of two contrasting factors--lung respiration and spontaneous motor activity--absent in anesthesized animals. Congestion and decongestion of the spleen are the physiological mechanisms compensating for variations in the level of oxygenation, an alternative to the "capillary recruitment" described by Poczopko and Burggren and Moalli in may amphibians that appears to be absent in newts. The newt spleen, known to play a lesser role in erythropoiesis and destruction of aged erythrocytes than that traditionally assigned to it is thus of primary importance in respiration.

The distribution of GABA-like-immunoreactive neurons in the brain of the newt, Triturus cristatus carnifex, and the green frog, Rana esculenta.

The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cells bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.

Evaluation of the carcinogenic risk of the phenoxyherbicide MCPA to an urodele amphibian

To evaluate the carcinogenic potential of the phenoxyherbicide 2-methyl-4-chlorophenoxy-acetic acid (MCPA) in the crested newt, Triturus cristatus carnifex, a long-term study has been carried out exposing the animals by the percutaneous route. Two hundred adult newts were divided into one control and three experimental groups of 20 females and 30 males each. The control group was kept in tap water and the experimental groups were kept for 4 days a week in an aqueous solution of Agroxone 3, a commercial formulation of MCPA, at concentrations equivalent to 100, 200, and 400 ppm of the active ingredient. Treatment was continued for 1 year, after which all the animals were kept under observation for approximately another year. Surviving female newts were killed 22–24 months after the beginning of experimentation, whereas the male newts were killed after 28 months, at the end of 18 weeks of exposure to the tumor promoter 12- O-tetradecanoylphorbol-13-acetate. Under experimental conditions, there was no carcinogenic activity of MCPA. Putative preneoplastic nodules of the liver and tumor-like lesions of the lower jaw were occasionally observed among the animals that survived more than 22 months after the beginning of experimentation. However, no significant differences in frequency between control and experimental groups were found.

Sites and trend of erythropoiesis in anemic, normal, and splenectomized newts.

Newts, Triturus cristatus carnifex (Laurenti), were anesthetized by submersion in 2% chlorbutol in tap water for 15 min, splenectomized and then rendered totally anemic two months later by treatment with acetylphenylhydrazine (APH) diluted in their tanks (25 mg/liter for 36 h, changing the solution every 12 h). In the 14 weeks following hemolysis, erythron restoration occurred with the same intermittence as it did in whole animals rendered anemic by APH treatment: Beginning the second week the red blood cell count progressively increases for about one month, followed by a period of stasis which lasts about three weeks, then by a new increase, and then by a final period of stasis. Histological examination shows that erythropoietic activity occurs partly in the circulating blood and partly in erythroblasts nestled in the crypts between the muscular trabeculae of the ventricle as well as in the atrial walls. These cells, which are not part of the freely circulating elements in the blood stream, become very abundant in both whole and splenectomized anemic newts but are also present in normal animals. Newts, thus, have three sites for erythropoiesis: the spleen, the blood stream, and the heart. The other components compensate for the elimination of the spleen without determining any lack of, or delay in, erythropoietic response.

Forebrain Development in Control and Hypothyroid Larvae ofTriturus Cristatus Carnifex

Some cytological and developmental aspects of the forebrain gray matter were studied in the control and the hypothyroid larvae of Triturus cristatus carnifex. The mitotic cell percentages in the periventricular germinal layer, the fusiform shape of the recently divided neuronal cells and the transitory gray matter cleavage, are characteristic of the immature status of the central nervous system (CNS) in this species. The hypothyroidism obtained during two months of thiourea treatment slows down the development in all the forebrain areas of the experimental larvae. Nevertheless, the nucleus olfactorius anterior and the corpus striatum, which have a longer mitotic activity with respect to the other neuronal structures, are more deeply affected by thyroid atrophy. Thyroid activity and forebrain maturation are discussed on the basis of the literature and our data with regard to their influence on behavior and evolution.

Microcythemia from anemic hypoxia and normal erythropoiesis in the newt.

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.

Periodic changes in the organs involved in the erythropoiesis of anemic newts.

In Triturus cristatus carnifex (Laurenti) newts rendered totally anemic by treatment with acetylphenylhydrazine (APH) diluted in their tank water (25 mg/liter for 48 hours, with four changes) the recovery of erythron occurs through periodic cycles of mitotic activity in the erythropoietic tissue. These cycles determine a marked increase in blood erythrocyte concentration at regular intervals of about 1 month. The consequence of this trend is the alternation of ferritin and hemosiderin accumulation phases during periods of stasis with iron mobilization phases during periods of erythropoietic activity, which is particularly evident in the Kupffer cells of the liver. Iron mobilization and erythropoietic activity are strictly related to the periodic hypertrophy of some Bowman's capsule cells in the renal corpuscle, which were previously denominated "lactate sensitive cells" (LSC). The histochemistry, location, and behavior of LSC indicate that they are probably the site of erythropoietin production in the newt.

Intermittent erythropoiesis in anemic newts.

A group of 88 newts, Triturus cristatus carnifex (Laurenti), was rendered totally anemic by administering acetylphenylhydrazine (APH) in the breeding water for 48 h at a concentration of 25 mg/liter. The course of erythron restoration was followed for 5 months, sacrificing four specimens per week and analyzing the blood and spleen hemopoietic tissue. The return to the normal values of the red blood cell count occurred through marked increases in concentration at fairly regular intervals, which is best explained by a discontinuous, rhythmic erythropoiesis. This fact is strictly correlated with the intermittent mitotic activity observed in the spleen and with the periodic appearance of large quantities of immature elements in the blood smears. The APH-induced synchronization of newt erythropoietic activity revealed the approximate length of each erythropoietic cycle to be 4 to 5 weeks and the erythropoietic life span to be 50 to 60 days.

Toxicity of 2-methyl-4-chlorophenoxyacetic acid to the adult crested newt

The toxicity of the herbicide Agroxone 3, a commercial formulation of the sodium salt of 2-methyl-4-chlorophenoxyacetic acid (MCPA), in the adult newt (Triturus cristatus carnifex) was tested after percutaneous exposure. At the concentration of 3200 ppm, the LT50 values were 17 and 21 hr for male and female newts. At 1600 ppm, the LT50 values were 35 and 45.5 hr. At 800 ppm, most of the animals were still alive after the 3 months of experimentation. There were no deaths at 400 and 200 ppm. Severe neuromuscular disorders were observed in animals exposed to lethal concentrations.