Control of renal function in larvalAmbystoma tigrinum

Summary Renal clearance methods were used to examine several factors which may be involved in control of urine flow in larvalAmbystoma tigrinum. Arginine vasotocin (10 ng/g) reduced both urine flow and GFR (–30%); this was reversed with mesotocin (5 ng/g). Adaptation to isosmotic media reduced GFR from 0.156 ml/10 g·h to 0.057. Neither hypophysectomy nor treatment with mesotocin altered this response. The adrenergic blocking agents propranolol and phentolamine did not reverse the antidiuresis in isosmotic medium-adapted larvac. Ten percent volume expansion with 50% and 100% Ringer's solution increased GFR in isosmotic medium-adapted animals. Hypophysectomy prevented this increase in GFR. Ten percent volume expansion with 200% Ringer's solution decreased GFR in tapwater adapted larvae; however, 20% volume expansion with 200% Ringer's increased GFR. Both volume and concentration of extracellular fluid appear to be important in control of GFR. Both arginine vasotocin and mesotocin may be involved in these responses, however other factors must also play a role.Abbreviations AVT arginine vasotocin - ECF extracellular fluid - GFR glomerular filtration rate - MT mesotocin     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma tigrinum tigrinum

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.     

Autoradiographic analyses of35S-sulfate uptake in regenerating limbs of larvalAmbystoma

Summary Autoradiographic and histochemical techniques were used to determine whether chondrocytes continue to synthesize chondroitin sulfate or closely related compounds during morphological dedifferentiation of these cells in regenerating limbs of larvalAmbystoma. Forelimbs were amputated either through the mid-diaphysis or the distal epiphysis of the humerus and each animal was subsequently injected with Na₂ ³⁵SO₄ at an appropriate stage of regeneration. Incorporation of the isotope and metachromatic staining responses were used as indices of cell specialization.In autoradiographs of unamputated limbs, epiphyseal chondrocytes exhibited moderate sulfate incorporation, whereas isotope uptake was slight in diaphyseal regions. Accordingly, in early stages of regeneration, limbs amputated through the diaphysis showed a low level of sulfate incorporation by cartilage-derived cells; since these cells dispersed during blastema formation, they were not identifiable in later stages. When limbs were amputated through the epiphysis, the matrix here underwent slow dissolution and epiphyseal-derived chondrocytes and their progeny consequently remained identifiable as they contributed to the blastema. These cells continued to exhibit isotope uptake, even during early and middle stages of regeneration —results which support the idea of tissue-specific regeneration of cartilage.Further inspection of the stained autoradiographs revealed that in addition to chondrocytes and blastema cells derived from chondrocytes, fibroblast-like cells located lateral to the limb skeleton and seemingly derived from muscle or muscle-associated cells also exhibited a moderate label during certain stages in the restoration of the limb. In several respects isotope incorporation and related metachromatic responses by these two types of cells during blastemal and early redifferentiating stages of regeneration were seen to parallel results reported in the literature of histochemical and autoradiographic studies of differentiating chick fimb buds. These observations, which may be added to previous analogies concerning developing and regenerating limbs, suggest a similar mechanism of cytodifferentiation in the two systems. The possibility is also considered that the observed isotope uptake by cells of non-cartilaginous origin may indicate the synthesis of sulfated glycosaminoglycans which alfect cell interactions during the regenerative processes.A portion of a dissertation submitted to the University of New Hampshire in partial fulfillment of the requirements for the degree of Doctor of Philosophy.NASA Predoctoral Trainee during the course of this work.The authors wish to thank Nr. Carl Paulitz for his assistance in photography of the autoradiographs.     

Kinetics of tongue projection in Ambystoma tigrinum: Quantitative kinematics,muscle function,and evolutionary hypotheses

The projectile tongue of caudate amphibians has been studied from many perspectives, yet a quantitative kinetic model of tongue function has not yet been presented for generalized (nonplethodontid) terrestrial salamanders. The purposes of this paper are to describe quantitatively the kinnematics of the feeding mechanism and to present a kinetic model for the function of the tongue in the ambystomatid salamander Ambystoma tigrinum. Six kinematic variables were quantified from high-speed films of adult A. tigrinum feeding on land and in the water. Tongue protrusion reaches its maximum during peak gape, while peak tongue height is reached earlier, 15 ms after the mouth starts to open. Tongue kinematics change considerably during feeding in the water, and the tongue is not protruded past the plane of the gape. Electrical stimulation of the major tongue muscles showed that tongue projection in A. tigrinum is the combined result of activity in four muscles: the geniohyoideus, Subarcualis rectus 1, intermandibularis posterior, and interhyoideus. Stimulation of the Subarcualis rectus 1 alone does not cause tongue projection. The kinetic model produced from the kinematic and stimulation data involves both a dorsal vector (the resultant of the Subarcualis rectus 1, intermandibularis posterior, and interhyoideus) and a ventral vector (the geniohyoideus muscle), which sum to produce a resultant anterior vector that directs tongue motion out of the mouth and toward the prey. This model generates numerous testable predictions about tongue function and provides a mechanistic basis for the hypothesis that tongue projection in salamanders evolved from primitive intraoral manipulative action of the hyobranchial apparatus.     

The composition and function of the pulmonary surfactant system during metamorphosis in the tiger salamander Ambystoma tigrinum

Mammalian lungs secrete a mixture of surface-active lipids (surfactant), which greatly reduces the surface tension of the fluid coating the inner lung surface, thereby reducing the risk of collapse upon deflation and increasing compliance upon inflation. During foetal lung maturation, these lipids become enriched in the primary and active ingredient, a disaturated phopholipid. However, disaturated phospholipids exist in their inactive gellike form at temperatures below 37°C and thus are inappropriate for controlling surface tension in the lungs of many ectotherms. We examined the development of the composition and function of the surfactant system of the tiger salamander (Ambystoma tigrinum) during metamorphosis from the fully aquatic larva (termed stage I) through an intermediate air-breathing larval form (stage IV) to the terrestrial adult (stage VII). Biochemical analysis of lung washings from these three life stages revealed a decrease in the percentage of disaturated phospholipid per total phospholipid (23.03 versus 15.92%) with lung maturity. The relative cholesterol content remained constant. The increased level of phospholipid saturation in the fully aquatic larvae may reflect their generally higher body temperature and the higher external hydrostatic compression forces exerted on the lungs, compared to the terrestrial adults. Opening pressure (pressure required for initial lung opening) prior to lavage decreased from larval to adult salamanders (7.96 versus 4.69 cm H₂O), indicating a decrease in resistance to opening with lung development. Opening pressure increased after lavage in older aquatic (stage IV) larvae (5.36 versus 9.80 cm H₂O) and in the adults (4.69 versus 7.65 cm H₂O), indicating that the surfactant system in salamanders may have an antiglue function which prevents apposing epithelial surfaces from adhering together.Abbreviations bm body mass - Chol cholesterol - DSP disaturated phospholipid - PC phosphatidylcholine - PL phospholipid - postlav postlavage - prelav prelavage - P-V pressure-volume - RH relative humidity - T_b body temperature - USP unsaturated phospholipid - WL wet lung mass     

Evidence for multiple Pleistocene refugia in the postglacial expansion of the eastern tiger salamander,Ambystoma tigrinum tigrinum

Abstract Pleistocene glaciations were important determinants of historical migration and, hence, current levels of genetic diversity within and among populations. In many cases, these historical migrations led to the existence of disjunct populations of plants and animals. However, the origin and timing of arrival of these disjunct populations is often debated. In the current study, we identify potential refugia and estimate the timing of vicariance events of the eastern tiger salamander, Ambystoma tigrinum tigrinum , using mitochondrial sequence data. The results suggest a vicariant event 0.75–2 million years ago, separating the tiger salamanders to the east and west of the Apalachicola River Basin. East of the Appalachians, there appear to be multiple independent refugia with little migration among the remaining populations. In particular, populations along the Atlantic Coastal Plain were likely isolated in a coastal plain refugium in the Carolinas. Migrants from this refugium were the likely source of colonists for populations occupying previously glaciated areas along the northeastern Atlantic Coast. A second potential refugium occurs in the Blue Ridge Mountains of western Virginia. This refugium contains a disjunct population of the eastern tiger salamander, as well as a community of nearly 70 other disjunct plant and animal species. The tiger salamanders here have been isolated from other populations for 200,000–500,000 years. These results suggest that disjunct mountain populations of Coastal Plain species may have existed in situ throughout the Pleistocene in Appalachian refugia. Therefore, these disjunct populations are not of recent origin, but rather exist as relicts of a warmer, more widespread fauna and flora that is now restricted to the Coastal Plain.     

Eicosanoids in renal function

The major role of renal eicosanoid synthesis appears to be a protective one. In the cortex, prostaglandin synthesis minimises potential anoxic and ischaemic damage by vasodilatation. In the medulla, prostaglandin synthesis appears to stabilise the corticomedullary solute gradient and may play a role in cell volume regulation. Mono-oxygenase production at this site, by modifying blood flow and cellular active transport processes could again serve a protective function against anoxia and ischaemia. The release of erythropoietin also appears to be prostaglandin dependent. It is likely that leukotrienes released from inflammatory cells within the kidney will affect renal haemodynamics and capillary permeability as in other tissues.     

Adenohypophysial-thyroid activity of the tiger salamander, Ambystoma tigrinum, as a function of metamorphosis and captivity

The aim of this study was to determine the timing of adenohypophysial activation during metamorphosis of the tiger salamander, Ambystoma tigrinum. It consisted of two parts: 1) determination of plasma thyroid hormone concentrations and analysis of thyroid gland histology as a function of metamorphic stage and 2) analysis of the time-course of uptake of 125I by the thyroids during metamorphosis as an indicator of endogenous thyrotropin (TSH) levels. Significant increases in both triiodothyronine (T3) and thyroxine (T4) first were evident at the onset of metamorphic climax (stage II). Maximum levels of both hormones were not observed, however, until the completion of gill resorption (stage VII). No changes in thyroid histology were observed that could be unambiguously related to metamorphic transformation. The thyroids accumulated 125I in a slow but linear fashion in premetamorphic larvae (stage I). However, uptake exhibited a rapid peak during early climax (stage II), before maximum concentrations of thyroid hormones were observed. In addition, uptake was maintained above premetamorphic levels at stage VII, in conjunction with maximum levels of T4 and T3. Captivity alone produced a small but significant increase in plasma concentrations of T3. It produced no significant effect on either thyroid histology or uptake of 125I. These results indicate that adenohypophysial activation occurs rapidly and is maximal at the onset of metamorphic climax.     

Incidence and Control of Necrotic Leaf Mosaic Caused by Arabis Mosaic Virus in Lilium tigrinum splendens in the Netherlands

Necrotic mosaic on leaves and ring spots on bulb scales of Lilium tigrinum splendens, can be caused by arabis mosaic virus (AMV). Primarily infected bulbs can show spongy roots and large necrotic areas on creamy coloured bulb scales. Consecutive series of plants replanted for monthly periods in infested soil were mostly infected by AMV at a high rate (70%) throughout the year. Very low or undetectable numbers of Xiphinema diversicandatum, nematodes in soil dilution experiments infected lilies very efficiently (50–70 %). In general, soil disinfestation with dichloropropene, dazomet, methylbromide, and other disinfectants were variably fairly effective, particularly when yellow crocus among which couch was abundant, was previously grown for two years. The influx of AMV infected material into the soil was assumed to increase the number of AMV-carrying nematodes, and may be one cause of the failure of soil disinfestation. A survey of AMV infested soil in lily-growing regions in The Netherlands indicated its occurrence in a few fields only. Complex control measures applicable under growers' conditions, in addition to the variably effective soil disinfestation, are indicated.     

Development of renal function

The mammalian metanephric kidney develops following a general principle of organogenesis of epithelial organs, i.e., along the tree-like structure of an arborizing ductal system (the ureteric bud and cortical collecting duct). In parallel, the proximal portions of the uriniferous tubule develop by mesenchymal-to-epithelial transition of the neighbouring mesenchyme. On one hand, vectorial transport systems in nephrogenesis should be functional at the onset of glomerular filtration in any of the newly formed nephron generations to prevent loss of salt, water and metabolites. On the other hand, developing nephron epithelia must serve the needs of organ-formation such as cell proliferation and fluid-secretion for morphogenic purposes. This review intends to summarize current data and concepts on the development of renal epithelial functions with an emphasis on ion channels. Current model systems are introduced, such as ureteric bud cell monolayer culture, in vitro nephron culture, HEK293 cell culture, and the dissection of tubular cells for direct analysis. The current data on the developmental expression and functions of ENaC Na⁺ channels, the CFTR, ClC-2 Cl^ndash; channels, L-type Ca²⁺ channels, P2 purinoceptors, and the Kir6.1/SUR2, ROMK (Kir1.1), and Kv K⁺ channels are presented.     

Experimental analysis of character coupling across a complex life cycle: Pigment pattern metamorphosis in the tiger salamander,Ambystoma tigrinum tigrinum

Developmental relationships among characters are expected to bias patterns of morphological variation at the population level. Studies of character development thus can provide insights into processes of adaptation and the evolutionary diversification of morphologies. Here I use experimental manipulations to test whether larval and adult pigment patterns are coupled across metamorphosis in the tiger salamander, Ambystoma tigrinum tigrinum (Ambystomatidae). Previous investigations showed that the early larval pigment pattern depends on interactions between pigment cells and the lateral line sensory system. In contrast, the results of this study demonstrate that the major features of the adult pigment pattern develop largely independently of both the early larval pattern and the lateral lines. These results suggest that ontogenetic changes that occur across metamorphosis decouple larval and adult pigment patterns and could thereby facilitate independent evolutionary modifications to the patterns during different stages of the life cycle. J. Morphol. 237:53–67, 1998. © 1998 Wiley-Liss, Inc.     

Acceleration of Ambystoma tigrinum metamorphosis by corticotropin-releasing hormone

Previous work of others and ours has shown that corticotropin-releasing hormone (CRH) is a positive stimulus for thyroid and interrenal hormone secretion in amphibian larvae and that activation of CRH neurons may mediate environmental effects on the timing of metamorphosis. These studies have investigated CRH actions in anurans (frogs and toads), whereas there is currently no information regarding the actions of CRH on metamorphosis of urodeles (salamanders and newts). We tested the hypothesis that CRH can accelerate metamorphosis of tiger salamander (Ambystoma tigrinum) larvae. We injected tiger salamander larvae with ovine CRH (oCRH; 1 microg/day; i.p.) and monitored effects on metamorphosis by measuring the rate of gill resorption. oCRH-injected larvae completed metamorphosis earlier than saline-injected larvae. There was no significant difference between uninjected and saline-injected larvae. Mean time to reach 50% reduction in initial gill length was 6.9 days for oCRH-injected animals, 11.9 days for saline-injected animals, and 14.1 days for uninjected controls. At the conclusion of the experiment (day 15), all oCRH-injected animals had completed metamorphosis, whereas by day 15, only 50% of saline-injected animals and 33% of uninjected animals had metamorphosed. Our results show that exogenous oCRH can accelerate metamorphosis in urodele larvae as it does in anurans. These findings suggest that the neuroendocrine mechanisms controlling metamorphosis are evolutionarily conserved across amphibian taxa.