Photoperiod,stress and the distribution of leukocytes in the peripheral blood ofNotophthalmus viridescens

Summary Adult newts,Notophthalmus viridescens, were maintained for 8 days at a constant temperature of 11.0±0.5 °C. In one series, the control animals were kept in constant darkness (DD), while the experimental newts were exposed to alternating 12-hour periods of light and dark (LD). In a second series, controlNotophthalmus lived in DD, and experimental animals lived in constant light (LL). In both series, the newts were sacrificed on the ninth day when blood smears were prepared. Differential counts of the leukocytes of animals that lived under the LD regimen were the same as those of controlNotophthalmus (Table 1). However, in newts that were maintained in LL, the neutrophils increased and the lymphocytes decreased relative to those types of cells in the controls (Table 1). Those changes indicate that continuous light constitutes stress for this species.Supported in part by a grant from the Committee on Research, Travel and Sabbatical Leaves, Colby College     

Differential Kin Discrimination by Red-spotted Newts (Notophthalmus viridescens) and Smooth Newts (Triturus vulgaris)

In organisms lacking parental care or when eggs of more than one female are layed together, parents preferentially tend to avoid eating their own offspring. In some species of newts (Caudata: Salamandridae), there is no parental care and cannibalism of eggs and larvae occurs when philopatric adults remain in the pond throughout the breeding season where eggs and larvae develop. Kin discrimination by both adult newts and larvae would be expected to occur during the breeding season as it would enable individuals to benefit from the nutrients obtained from eating eggs and larvae while they avoid eating close relatives. The effects of kinship on cannibalism of eggs and larvae were examined in two species of newts. In separate, but similar, paired behavioral trials adult female red-spotted newts, Notophthalmus viridescens, and smooth newts, Triturus vulgaris, were allowed to consume their own egg or an egg from another female. Female red-spotted newts, but not smooth newts, preferred to eat eggs of other females rather than their own, and overall smooth newts were ‘reluctant’ to eat conspecific eggs. To test for ontogenetic shifts in discrimination abilities, additional experiments were performed with adult female red-spotted newts paired with their own larvae and a larva from another female. In addition, the effects of kinship on the attraction or repulsion of larvae was also studied in red-spotted newts. Females showed no significant discrimination abilities during acts of cannibalism on larvae. Larvae spent similar amounts of time with siblings and non-siblings. These results suggest that the ability of adult newts to discriminate among eggs varies between species and that the elicitation of a kin-discrimination response may be context-dependent. For red-spotted newts, there was no evidence of discrimination abilities within and between other life-history stages.     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

Interactions of bullfrog tadpole predators and an insecticide: predation release and facilitation

The effect of a contaminant on a community may not be easily predicted, given that complex changes in food resources and predator-prey dynamics may result. The objectives of our study were to determine the interactive effects of the insecticide carbaryl and predators on body size, development, survival, and activity of tadpoles of the bullfrog (Rana catesbeiana). We conducted the study in cattle tank mesocosm ponds exposed to 0, 3.5, or 7.0 mg/l carbaryl, and no predators or two red-spotted newts (Notophthalmus viridescens), bluegill sunfish (Lepomis macrochirus), or crayfish (Orconectes sp.). Carbaryl negatively affected predator survival by eliminating crayfish from all ponds, and by eliminating bluegill sunfish from ponds exposed to the highest concentration of carbaryl; carbaryl exposure did not effect survival of red-spotted newts. Because crayfish were eliminated by carbaryl, bullfrogs were released from predation and survival was near that of predator controls at low concentrations of carbaryl exposure. High concentrations of carbaryl reduced tadpole survival regardless of whether predators survived carbaryl exposure or not. Presence of crayfish and newts reduced tadpole survival, while bluegill sunfish appeared to facilitate bullfrog tadpole survival. Presence of carbaryl stimulated bullfrog tadpole mass and development. Our study demonstrates that the presence of a contaminant stress can alter community regulation by releasing prey from predators that are vulnerable to contaminants in some exposure scenarios.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

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.     

Magnetic compass orientation in the Eastern red-spotted newt (Notophthalmus viridescens)

Summary Laboratory tests were carried out to examine the orientation behavior of adult Eastern red-spotted newts (Notophthalmus viridescens) to earth-strength magnetic fields. Groups of 30 to 40 newts were housed in water-filled, all-glass aquaria with an artificial shoreline at one end. The aquaria were located in a greenhouse or outdoors adjacent to the laboratory building, and aligned on either the magnetic north-south or east-west axis. Tests were carried out in an enclosed indoor arena. Newts were tested in four horizontal alignments of the magnetic field: the ambient magnetic field (magnetic north at North) and three altered fields (magnetic north rotated to East, South or West). Data were analyzed after pooling the magnetic bearings from all four conditions in such a way as to retain the component of the newts' orientation that was a consistent response to the magnetic field. Elevation of training tank water temperature was used to increase the newts' motivation to orient in the direction of shore. Newts exposed to a training tank water temperature of 33–34 °C just prior to testing exhibited consistent unimodal magnetic compass orientation. The direction of orientation was altered predictably by changing training tank alignment and location relative to the laboratory building. The results provide the first evidence of a strong, replicable magnetic compass response in a terrestrial vertebrate under controlled laboratory conditions. Further, the present study demonstrates that the Eastern newt is able to learn a directional response relative to the earth's magnetic field.     

Sexual interactions in red-spotted newt populations

Two alternative reproductive tactics (amplexus and spermatophore transfer interference) and three sexual interference tactics (spermatophore transfer interference, pseudofemale behaviour, amplexus interference) are described for male red-spotted newts, Notophthalmus viridescens. The two reproductive tactics yield equal insemination rates for males displaying these behaviours. The three sexual interference tactics decrease the potential fitness of amplectic males either directly, by displacing them from receptive females and/or duping them into fruitless spermatophore depositions, or indirectly, by increasing the time spent in amplexus with no concomitant increase in probability of successful insemination.     

Parasite age-intensity relationships in red-spotted newts: does immune memory influence salamander disease dynamics?

Acquired immune memory in vertebrates influences transmission and persistence of infections, with consequences for parasite dynamics at both the individual and population levels. The potential impact of acquired immunity is of particular interest for salamanders, whose acquired immune systems are thought to be less effective than those of frogs and other tetrapods. One way to examine the importance of acquired immunity to parasite dynamics at the population level is by examining the relationship between host age and parasite infection intensity. Acquired immunity reduces infection rates in older animals, causing decreased parasite intensity in older age classes and leading to curvilinear age-intensity relationships for persistent parasites and convex age-intensity relationships for transient parasites. We used age-intensity relationships to look for the signature of acquired immunity for 12 parasite taxa of red-spotted newts (Notophthalmus viridescens), using data from a 2-year parasitological survey of six newt populations. We estimated ages from snout-vent length (SVL) based on the relationship between SVL and skeletochronologically-derived ages in a subset of newts. We found evidence of acquired immunity to two parasite taxa, bacterial pathogens and the protist Amphibiocystidium viridescens, whose convex age-intensity relationships could not be easily explained by alternative mechanisms. Our results suggest that the acquired immune response of newts is sufficient to influence the dynamics of at least some parasites.     

A scanning electron microscopic and quantitative histologic description of lens regeneration in the newt,Notophthalmus viridescens

Adult newts (Notophthalmus viridescens) were lentectomized and at intervals from 4 to 21 days after lentectomy iridocorneal complexes from these animals were examined by scanning electron microscopy to allow a full appreciation for the shape of the regenerating lens. Until around day 12 after lentectomy the posterior surface of the iris is covered by a dense mat of fibrous material which cannot be removed without damage to the iris and which obscures the events of cytoplasmic shedding. The regenerate becomes visible first around stage IV (day 12). A small but clear groove demarcates the regenerate from the rest of the iris. As regeneration progresses there is a marked reduction in debris on the iris surface and the regenerate appears as a U-shaped thickening occupying about one-third of the dorsal half of the iris. During later stages (VI–X) the regenerate protrudes into the pupil inferiorly and posteriorly towards the retina, but does not encroach laterally on the remaining pigmented iris tissue. Prior to secretion of the lens capsule the outline of individual cells is visible on the surface of the regenerate and some regenerates exhibit a prominent dimple on their posterior aspects. Following secretion of the capsule the surface of the regenerate becomes smooth. Quantitative studies show that volume and maximum section area of the regenerate are both more strongly correlated with developmental stage of regeneration than with time after lentectomy.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

The histology of inhibition of limb regeneration in the newt, Triturus, by actinomycin D

Adult newts, Triturus viridescens, were treated with from 1.0–10.0 μg/g body weight of actinomycin D one day before amputation of both forelimbs. Mean survival times ranged from over 50 days in newts treated with 1.0 μg/g to 13.2 days in animals given 10.0 μg/g body weight of actinomycin. Low doses little altered the course of regeneration, but animals treated with over 2.0 μg/g never formed blastemas. In another series, animals were given doses of 2.5 μg/g body weight of actinomycin D at intervals from 14 days before to 30 days after amputation. It was found that certain signs of toxicity (loss of equilibrium) are related to the time of administration of the drug whereas others (hemorrhage into the limb stumps) are restricted to a definite phase of the regenerative process. Early administration of actinomycin completely inhibits regeneration whereas later treatment results in a considerably lessened effect. The postamputational stages which are basically destructive in nature are not noticeably affected by actinomycin D, but the phases of dedifferentiation, blastema formation and redifferentiation are strongly inhibited.     

Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb

Dorsal iris from the eyes of adult Notophthalmus viridescens was transplanted into the blastema of regenerating limbs, subcutaneously in the limb or shoulder region, into the dorsal fin of larval newts and into the hindbrain of larval Ambystoma maculatum. The iris implants into the blastema regenerated lens vesicles or lenses with fibers in 40–75% of the cases. Multiple lenses were found in a few instances. No lenses developed from iris implants into the dorsal fin. Twenty percent of subcutaneous implants of iris formed lenses or lens vesicles, but lens regeneration from implants into the brain occurred only rarely. Denervation of the limb at the time of iris transplantation into the blastema greatly reduced the number of lenses regenerated. Studies on nerve fiber distribution in dorsal fin, subcutaneous areas, and denervated and innervated regenerating limbs, using the Bodian method, showed a general correlation between density of nerve fibers in the implant site and the incidence of lens regeneration from iris implants into that site. These results provide some evidence for a trophic action of nerve fibers on lens regeneration from the iris.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Amphibian skin may select for rare environmental microbes

Host-microbe symbioses rely on the successful transmission or acquisition of symbionts in each new generation. Amphibians host a diverse cutaneous microbiota, and many of these symbionts appear to be mutualistic and may limit infection by the chytrid fungus, Batrachochytrium dendrobatidis, which has caused global amphibian population declines and extinctions in recent decades. Using bar-coded 454 pyrosequencing of the 16S rRNA gene, we addressed the question of symbiont transmission by examining variation in amphibian skin microbiota across species and sites and in direct relation to environmental microbes. Although acquisition of environmental microbes occurs in some host-symbiont systems, this has not been extensively examined in free-living vertebrate-microbe symbioses. Juvenile bullfrogs (Rana catesbeiana), adult red-spotted newts (Notophthalmus viridescens), pond water and pond substrate were sampled at a single pond to examine host-specificity and potential environmental transmission of microbiota. To assess population level variation in skin microbiota, adult newts from two additional sites were also sampled. Cohabiting bullfrogs and newts had distinct microbial communities, as did newts across the three sites. The microbial communities of amphibians and the environment were distinct; there was very little overlap in the amphibians'' core microbes and the most abundant environmental microbes, and the relative abundances of OTUs that were shared by amphibians and the environment were inversely related. These results suggest that, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment.     

Thermal influence on metabolic rates and a bioenergetic budget for Notophthalmus viridescens from southwestern Massachusetts

Notophthalmus viridescens has been reported to overwinter on land in southwestern Massachusetts, whereas these newts hibernate in water in southwestern Ohio. Aquatic and terrestrial metabolic rates of newts from Massachusetts were measured at different exercise speeds and acclimation temperatures in order to better understand their seasonal energetic budgets. Oxygen uptake at 25°C increased with increased swimming and walking speeds and reached a plateau at speeds of 60 and 90 cm/min, whereas at 5°C, oxygen consumption linearly increased with swimming speeds. Aerobic transport costs of the newts thus decreased with increased locomotor speeds at 25°C but remained unchanged when the newts were exercised in water at 5°C. Anaerobic metabolic rates of the newts on land were little affected by acclimation temperature but also increased linearly with walking speeds at both 5°C and 25°C. Anaerobiosis contributed most of the energy for the locomotion of the newts. These newts stored an average of 12 mg lipd/g body mass, which could apparently support their survival at 5°C for 46 days without food on land but only for 18 days in water. These calculations, based on measured metabolic rates and energy reserves, support field observations of red-spotted newts hibernating on land in Massachusetts.     

Changes in the blood of newts,Notophthalmus viridescens,following the administration of hydrocortisone

Summary Adult newts,Notophthalmus viridescens, were injected with suspensions of hydrocortisone acetate (experimentais) or with distilled water (controls). Forty-eight and 72 hours after treatment, blood smears were prepared, and differential counts of leucocytes were made for the experimental and control animals. At 48 hours, the distributions of neutrophils, eosinophils, basophils, monocytes and lymphocytes were much the same in the two groups of newts (Table 1). However, by 72 hours after injection, increases in neutrophils and decreases in lymphocytes were obvious in the animals which had received hydrocortisone. Such changes were not seen in the controls (Table 2). The changes in the distribution of the white cells seen 72 hours after treatment are very similar to those known to occur in mammals treated with adrenal steroids and to those described earlier in two species of frogs injected with hydrocortisone. Details of some differences in the responses of the amphibians are discussed.Supported by National Science Foundation COSIP grant, GY-7661, to Sweet Briar College.     

Spontaneous preferences for magnetic compass direction in the American red-spotted newt, <Emphasis Type="Italic">Notophthalmus viridescens</Emphasis> (Salamandridae,Urodela)

In addition to other sensory modalities, migratory vertebrates are able to use the earths’ magnetic field for orientation and navigation. The magnetic cue may also serve as a reference for other orientation mechanisms. In this study, significant evidence is shown that, even in darkness, newts (Notophthalmus viridescens, Salamandridae) spontaneously align according to the natural or to the deviated earth’s magnetic field lines, thereby demonstrating a magnetic compass sensitivity. All newts preferred compass directions close to east or west or chose the E/W axially and hence sought to maintain a specific angle or axis relative to the magnetic field vector. Such an active alignment is considered an essential precondition for magnetic orientation. When the horizontal magnetic vector was experimentally compensated, animals became disoriented. We infer that the animals have either learned the preferred magnetic direction/axis individually or that these choices are innate and could even be seasonally different as in migrating birds. It is still an unanswered question as to how and where the physical and physiological mechanisms of magnetic transduction and reception take place. The visual system and other light-dependent (radical pairs) mechanisms alone are often claimed to be in function, but this must now be reconsidered given the results from animals when deprived of light. The results may therefore point to putative receptor mechanisms involving magnetite elements in specialized magneto-receptors.     

Osmotic stress,ACTH and the white blood cell picture in newts,Notophthalmus viridescens

Summary Adult newts,Notophthalmus viridescens, were given single injections of 0.04 I.U. of bovine ACTH or were immersed in 2% NaCl for 2 hours. In both the corticotrophin-treated and the osmotically-stressed newts, increases in neutrophils and decreases in lymphocytes were obvious and significant. Neither condition was observed in the controls (Tables 1 and 2). The changes seen in the peripheral blood of the experimentalNotophthalmns were very much like those reported earlier for animals of the same species which had received injections of the adrenal steroid, hydrocortisone. Using these lines of evidence, it is argued that neutrophilia and lymphopenia in amphibians could result from a stress anterior pituitary steroidogenic tissue-system of reactions which has been proved to effect similar changes in the blood of stressed mammals.Supported in part by National Science Foundation grant, GY-7661, to Sweet Briar College.     

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.