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.     

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.     

Characteristics of newt breeding sites

Breeding site characteristics have been studied for the three species of newt that occur in Britain, the Palmate ( Triturus helveticus (Razoumowski)), Smooth ( T. vulgaris (L.)) and Warty ( T. cristatus (Laurenti)). The Warty newt was seldom found in the absence of the much commoner Smooth newt, but seemed to prefer sites that were relatively large and deep and that had a high proportion of open water surface. All three species tended to breed in ponds having abundant aquatic vegetation. Smooth newts, unlike Palmate newts, were rarely encountered in water with pH <6. The Smooth newt tended to be found in water with relatively high concentrations of metals, while the reverse was true for the Palmate newt. Over Britain, Smooth and Warty newts are relatively less common in soft water areas, while the Palmate is less common in hard water areas. Possible reasons for these associations are discussed.     

Tolerance, limb regeneration and collagen fibrogenesis under high oxygen concentrations by the adult newt, Diemictylus viridescens

Adult newts placed in an atmospheric environment of 85% oxygen, saturated humidity, and at a temperature of 20 ± 1°C survived particularly well a 44-day test period. They did not succumb to “oxygen toxicity” as has been frequently reported for other vertebrate species. Having established the newt's tolerance of high oxygen atmosphere, the effect of oxygen on growth and development in the regenerating newt limb was investigated. Under the atmospheric conditions described above, and under 92% oxygen, the regeneration of adult newt limbs appeared to be retarded during the first 25 days after amputation when compared with regenerating limbs of control animals kept under a normal atmosphere of 21% oxygen (air). Thereafter, little or no difference could be discerned between the regeneration of experimental and control limbs. It is known that molecular oxygen participates directly in the hydroxylation of proline to hydroxyproline in the synthesis of collagen. Sectioned regenerates stained specifically for collagen were examined to determine if collagen synthesis was induced in experimental animals. Two regeneration-inhibited limbs of oxygenated newts showed cicatrical repair of the apical limb stump 25 days after amputation. However, the majority of the experimental animals revealed no obvious increase in collagen fibers. These results contraindicate any marked “oxygen toxicity” affecting the life of the newts, or regeneration of their limbs. It is suggested that a change in collagen fiber type might have been induced by the high-oxygen atmosphere. Investigations to test this hypothesis are currently underway.     

Thyroid and hypoxic stress in the newt Triturus carnifex

When specimens of the newt Triturus carnifex, under anaesthesia by submersion in a 0.2% chlorbutol solution for 25 min, are isolated in a respiratory chamber at 18 degrees C containing water with only 1.3 ppm of oxygen, they consume the oxygen completely in about 3 hr, but they can stay alive for many more hours and wake up with no apparent exterior consequences. Hypoxia induces rapid onset of hepatic steatosis and melanosis, as well as a controlled haemolytic process involving a pool of red blood cells of the same order of size as that held as a reserve in the spleen by animals in an aerial habitat. At the origin of the phenomena is an intense response by the hypophysis, histologically detectable 1 hr from the onset of treatment and confirmed 2 hr later by a highly significant increase in the plasma thyroidstimulating hormone (TSH) concentration compared with the controls (41.5 +/- 13.7 microU/L vs. 15.5 +/- 6.2; P < 0.005). The thyroid follicles react by reabsorbing their colloid, but instead of an increase in the plasma free T3 and T4 concentrations, fT3 falls significantly (1.5 +/- 0.3 pg/mL vs., the 2.4 +/- 0.7; P < 0.05), whereas fT4 remains stationary (4.0 +/- 0.5 pg/mL vs. 4.6 +/- 0.8; N.S.). After 6 hr, the plasmatic TSH concentration is still higher than in the controls (27.0 +/- 3.0 microU/L vs. 15.5 +/- 6.2; P < 0.05), whereas fT3 and fT4 remain stable (1.5 +/- 0.3 and 4.4 +/- 0.5 pg/mL, respectively). If T3 or T4 labelled with 125I is administered prior to hypoxia, after 6 hr of treatment the radioactivity is found to be limited exclusively to the liver and kidney; the thyroid, gall bladder and gut result negative, and this does not agree with hypotheses of hormone inactivation by deiodination, sulphation or glucuronidation. This apparently peculiar endocrine path has not been observed in previous studies on hypoxia in vertebrates, because the experiments were always designed to analyse plasma hormone levels after at least 24 hr of hypoxia or during chronic treatments, losing the most interesting phases of the endocrine response. The possibility that the hypoxic newt possesses alternative or complementary metabolic pathways to anaerobic glycolysis to sustain steatogenesis and melanogenesis and maintain the same cardiac activity as the controls is briefly discussed.     

Replacement of a unique population of newts (<Emphasis Type="Italic">Taricha granulosa mazamae</Emphasis>) by introduced signal crayfish (<Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis>) in Crater Lake,Oregon

The signal crayfish (Pacifastacus leniusculus) was introduced to Crater Lake in 1915 and now threatens the local extinction of an endemic salamander, the Mazama newt (Taricha granulosa mazamae). More than a century after their introduction, crayfish have expanded in distribution to occupy nearly 80% of the lakeshore. Although newts remain in uninvaded areas, they are almost entirely absent in crayfish occupied areas. Abundance of benthic macroinvertebrates was dramatically reduced in locations with crayfish compared with areas of the lake where crayfish were absent. Isotopic signatures of newt and crayfish tissue confirm overlap in the diets of the two species and demonstrate their similar position in the Crater Lake food web. Mesocosm experiments conducted with newts and crayfish revealed that crayfish prey directly on newts, displace newts from cover, and generally alter newt behavior. Combined, this evidence suggests that further crayfish expansion likely will cause additional declines in newt abundance and distribution, and could lead to extinction of the unique population of newts in Crater Lake.     

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.     

Limitation of similarity and feeding habits in three syntopic species of newts (Triturus, Amphibia)

Differences in body size between both species and sex were observed to be close to Hutchinsonian ratios in an assemblage of three species of newts (Triturus italicus, T alpestris and T carnifex) in southern Italy According to feeding, differences in body size were not related with a strict food niche segregation The diet of larger newt included indeed all the prey eaten by smaller newts but showed new prey types Differences between species were more marked than between sexes of the same species, despite a strong sexual dimorphism in body size Larger newts foraged in a wider range of microhabitats and exhibited a greater diversity of foraging tactics Triturus italicus fed essentially on plankton and chironomids in shallow water at the banks of the pond Triturus alpestris foraged also in deeper water, particularly on lsopods in vegetation Prey fallen on the water surface constituted the main part of T carnifex diet According to recent data on newt evolution, speciation consisted in the reduction of body size Our data show that this process has been related with a specialization of the diet and of habitat use     

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.     

Impact of chaparral wildfire-induced sedimentation on oviposition of stream-breeding California newts (Taricha torosa)

We examined the effects of chaparral wildfire on stream-breeding California newts (Taricha torosa) in a 750-m stretch of a perennial Santa Monica Mountain stream (Los Angeles County). Detailed field surveys of 1992 and 1993 established the composition (run, riffle, pool) of this habitat and determined the oviposition sites of newts. We also quantified California newt egg mass density and estimated the density of newt adults. A chaparral wildfire burned the entire study site on 2 November 1993. Using the same methods, we collected field survey data in 1994 and 1996. Erosion following the 1993 wildfire produced major changes in stream morphology and composition. Pools and runs represented approximately 40–50% of pre-fire stream area. In the spring following the fire, the stream consisted of less than 20% run and pool. Pools that did remain were often smaller and shallower. The average density of adult California newts did not differ among years. The total number of newt egg masses observed in the spring after the fire was approximately one-third of egg mass counts from pre-fire surveys. Most California newt egg masses were laid in pools and runs; California newts prefer deeper slow-moving water. We conclude that fire-induced landslides and siltation have eliminated pools and runs, thus reducing the amount of habitat suitable for oviposition. Habitat alterations caused by fire likely account for the observed reduction of egg masses at the stream. Received: 11 June 1996 / Accepted: 18 December 1996     

Phosphorus and fluorine NMR examination of the anesthetized newt (Notopthalamus viridescens)

We have examined newts by 19F-NMR using the anesthetic halothane as a probe and in another set of experiments taken 31P-NMR spectra under similar conditions. The spectra were recorded from the animal's tail. The water soluble 31P-NMR signals point to little difference between anesthetized and unanesthetized newts except for the potential disappearance of two pools of inorganic phosphate in the anesthetized animals. The 19F spectra show two anesthetic populations in the tail which the phosphorus spectra suggest arise from populations of halothane in muscle and in lipid.     

The application of eDNA for monitoring of the Great Crested Newt in the UK

Current ecological surveys for great crested newts are time‐consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advantageous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Consequently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.     

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.     

The influence of temperature on diving behaviour in the alpine newt, Triturus alpestris

An aquatic lifestyle poses serious restriction to air-breathing animals in terms of time and energy spent during a dive cycle. The diving frequency increases with water temperature, therefore an ectotherm's time budget greatly depends on the thermal characteristics of the aquatic environment. Available data suggests that time costs caused by temperature-dependent dive frequency can be partially compensated for by adjusting the swimming speed and diving angle during dive cycle. We tested this prediction by examining the influence of temperature on the diving behaviour of the alpine newt, Triturus alpestris. The ascending speed and angle showed disparate patterns of temperature dependency, with a minor influence on travel duration. Surprisingly, at higher temperatures, the diving newts saved most of their time by restricting swimming activity in the water column during their return to the bottom and not by adjusting their ascending duration. Hence, aquatic newts have the capacity to reduce temperature-dependent time costs of aerial breathing primarily by behavioural modifications during the descending phase of the dive cycle.     

Dissociation between the circadian rhythm of locomotor activity and the pineal clock in the Japanese newt

The circadian locomotor activity rhythm of the Japanese newt has been thought to be driven by a putative brain oscillator(s) subordinate to the pineal clock. The existence of mutual coupling between the pineal clock and the brain oscillator(s) in vivo was examined. We covered the newt's skull with aluminum foil and simultaneously reversed the light-dark cycle, thereby allowing the pineal organ to be exposed to constant darkness while the rest of the animal was exposed to the reversed light-dark cycle. In control animals, whose heads were covered with transparent plastic, the rhythm of synaptic ribbon number in the pineal photoreceptor cells was entrained to the reversed light-dark cycle. Rhythms from newts whose heads were shielded, however, were similar to those observed in the unoperated newts kept under constant darkness. The locomotor activity rhythms of both head-covered animals and control animals were entrained to the reversed light-dark cycle. These data suggest that extrapineal photoreception can entrain the putative brain oscillator(s), but not the pineal clock. Thus, at least in an aspect of photic entrainment, there seems to be little or no mutual coupling between the pineal clock and the putative brain oscillator(s) in the circadian system of the Japanese newt.Abbreviations LD light-dark - DD constant darkness - SCN suprachiasmatic nucleus - SR synaptic ribbon     

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.     

Molecular genetic system for regenerative studies using newts

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.     

Functional equivalency of created and natural wetlands: diet composition of red-spotted newts (Notophthalmus viridescens viridescens)

Evaluating the adequacy of created wetlands to replace functions of lost natural wetlands is important because wetland mitigation is a major tool used to offset wetland losses. However, measurements such as vegetative cover and wildlife presence may not be evidence enough that created wetlands are functioning properly and thus, examining the ecology of wetland biota such as amphibians may be a more useful surrogate for function. Our objectives were to measure the diet composition of adult red-spotted newts (Notophthalmus viridescens viridescens) and compare the selection of prey by newts between created and natural wetlands. Newts were trapped during the spring and summer of 2009 and 2010, and the stomach contents of 149 newts were obtained with gastric lavage. Invertebrate prey availability was obtained within a 5 m radius of each captured newt. Selection of prey by newts was nonrandom, but was only minimally affected by wetland type. Both dietary breadth and prey selection were affected primarily by time of year, likely driven by temporal variation in invertebrate abundance. Our results suggest that the function of providing an adequate prey base for a generalist wetland predator such as the red-spotted newt is being fulfilled for the created wetlands that we examined.     

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.