Neotenic Salamander Research Articles

Anatomy and forebrain projections of the olfactory and vomeronasal organs in axolotls (Ambystoma mexicanum).

We examined the anatomy of the nasal cavity and forebrain in the axolotl (Ambystoma mexicanum) to determine whether the olfactory and vomeronasal systems are present in this neotenic aquatic salamander. The current study was motivated by two considerations: (a) little is known of the anatomy of the vomeronasal system in aquatic vertebrates, and (b) the presence of both olfactory and vomeronasal systems in larval amphibians has broad implications for the evaluation of these systems in vertebrates. From cresyl-violet-stained sections of snouts we determined that the nasal cavity of axolotls is much like that of terrestrial salamanders. The main chamber of the nasal cavity contains an olfactory epithelium, which is confined to grooves between longitudinal ridges of connective tissue covered in a nonsensory epithelium which lacks goblet cells. Using transmission electron microscopy, we found morphologically distinct olfactory receptor cells: many receptor cells terminate in microvillar dendrites, and fewer terminate in motile cilia with the 9 + 2 microtubule array typical of vertebrate olfactory receptor cells. The ciliated and microvillar cells occur in clusters with little intermingling. Horseradish peroxidase labeling revealed that axons of the olfactory receptor cells terminate in large glomeruli in the main olfactory bulb at the rostral end of the telencephalon. Lateral to the main chamber of the nasal cavity is a diverticulum that is entirely lined with a vomeronasal epithelium containing basal cells, microvillar receptor cells, sustentacular cells that lack specialized processes on the apical surface, and large ciliated cells that may function to move fluid across the vomeronasal epithelium. Unlike the olfactory epithelium, the vomeronasal epithelium lacks Bowman's glands. Using horseradish peroxidase, we determined that the axons of the vomeronasal receptor cells project to the accessory olfactory bulb, a distinct structure dorsal and caudal to the main olfactory bulb. The presence of both olfactory and vomeronasal systems in axolotls and other neotenic salamanders implies that both systems are pleiomorphic in larval amphibians; we therefore suggest that the vomeronasal system may not have originated as an adaptation to terrestrial life.

Anatomy and Forebrain Projections of the Olfactory and Vomeronasal Organs in Axolotls <i>(Ambystoma mexicanum)</i> (Part 2 of 2)

We examined the anatomy of the nasal cavity and forebrain in the axolotl <i>(Ambystoma mexicanum) </i>to determine whether the olfactory and vomeronasal systems are present in this neotenic aquatic salamander. The current study was motivated by two considerations: (a) little is known of the anatomy of the vomeronasal system in aquatic vertebrates, and (b) the presence of both olfactory and vomeronasal systems in larval amphibians has broad implications for the evolution of these systems in vertebrates. From cresyl-violet-stained sections of snouts we determined that the nasal cavity of axolotls is much like that of terrestrial salamanders. The main chamber of the nasal cavity contains an olfactory epithelium, which is confined to grooves between longitudinal ridges of connective tissue covered in a nonsensory epithelium which lacks goblet cells. Using transmission electron microscopy, we found morphologically distinct olfactory receptor cells: many receptor cells terminate in microvillar dendrites, and fewer terminate in motile cilia with the 9 + 2 microtubule array typical of vertebrate olfactory receptor cells. The ciliated and microvillar cells occur in clusters with little intermingling. Horseradish peroxidase labeling revealed that axons of the olfactory receptor cells terminate in large glomeruli in the main olfactory bulb at the rostral end of the telencephalon. Lateral to the main chamber of the nasal cavity is a diverticulum that is entirely lined with a vomeronasal epithelium containing basal cells, microvillar receptor cells, sustentacular cells that lack specialized processes on the apical surface, and large ciliated cells that may function to move fluid across the vomeronasal epithelium. Unlike the olfactory epithelium, the vomeronasal epithelium lacks Bowman's glands. Using horseradish peroxidase, we determined that the axons of the vomeronasal receptor cells project to the accessory olfactory bulb, a distinct structure dorsal and caudal to the main olfactory bulb. The presence of both olfactory and vomeronasal systems in axolotls and other neotenic salamanders implies that both systems are plesiomorphic in larval amphibians; we therefore suggest that the vomeronasal system may not have originated as an adaptation to terrestrial life.

Thyroid hormone receptors and iodothyronine deiodinases in the developing Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is a neotenous salamander that rarely undergoes anatomical metamorphosis, but can be induced to do so by administration of thyroxine (T 4). The neoteny appears to be due primarily to low levels of plasma T 4 secondary to a low rate of secretion of thyroid-stimulating hormone. However, other factors may also be involved. In anuran amphibia, metamorphosis is accompanied by alterations in thyroid hormone receptor concentration and marked changes in the activities of the iodothyronine deiodinase systems, all of which contribute to enhancing peripheral sensitivity to circulating T 4. The present study was designed to assess these functions in the tissues of the axolotl. Putative 3,5,3′-triiodothyronine (T 3) receptors were readily detected in axolotl red blood cells, and the receptor number (sites/nucleus) showed a developmental decline, comparable to that seen in anuran amphibia, as the larval cells with a high receptor number were replaced with adult cells with a low receptor number. Saturable T 3 nuclear binding sites were also detected in liver, but the receptor number was too low to quantitate. Using a sensitive RIA, T 4 and T 3 were below detectable levels in juvenile axolotls and T 4 was just detectable in some of the adults. Following injection of [ 125I]T 4, [ 125I]T 3 was detected in plasma and liver of adult, but not of juvenile, axolotls. Some 5′-deiodinase activity was consistently detected in preparations of larval skin, and in adult skin and gut, but it was too low to permit kinetic analyses. Activity was not increased following treatment with sufficient T 4 to induce anatomical metamorphosis. 5-Deiodinase activity was not detected in any tissue. The studies support the concept that the major cause of anatomical neoteny in the axolotl is an inadequate plasma T 4 concentration. They also suggest that axolotl tissues, unlike those of anuran amphibia, do not undergo some of the specific changes which would be expected to enhance the physiological response to the available T 4.

Cloacal anatomy of metamorphosed and neotenic salamanders

Ambystoma gracile is a polymorphic species, with some individuals attaining sexual maturity in the larval form. Such neotenic A. gracile retain larval characters in nonreproductive tissues. The cloacal anatomy of metamorphosed and neotenic individuals of both sexes was examined to determine the degree to which the larval state is reflected in reproductive structures. No difference in anatomy or onset of development of cloacal glands exists between metamorphosed and neotenic salamanders; thus, individuals should be capable of interbreeding. One autotriploid male showed few signs of cloacal gland development despite well-developed somatic growth, which supports the hypothesis of delayed maturity or sterility in male polyploid salamanders.

Adenylate cyclase uncoupled β-adrenergic receptors in salamander proximal tubules

The isolated perfused proximal tubule of the neotenic salamander Ambystoma tigrinum responds with either a hyperpolarization or depolarization of both the basolateral cell membrane and transepithelial potentials following the addition of 10(-5) M isoproterenol to the bath superfusate. Both responses were blocked by 10(-6) M propranolol but neither response was mimicked by 10(-4) M cAMP. beta-Adrenergic binding studies of individual microdissected proximal tubules using (-)-[3H]CGP-12177 as a hydrophyllic radioligand and (+/-)-timolol (0.1 mM) as the displacer drug revealed two distinct populations of proximal tubules possessing either low (KD = 153.8 nM; Bmax = 110.2 fM/mm) or high affinity (KD = 12.0 nM: Bmax = 3.9 fM/mm) binding characteristics. Competition studies indicated that the bound (-)-[3H]CGP-12177 behaved as a typical beta-adrenergic ligand, being displaced by (-)-isoproterenol but not by (+)-isoproterenol or (-)-phenylephrine. However, neither appeared to be coupled to the adenylate cyclase system. These data suggest the presence of functional beta-adrenergic receptors that do not appear to be coupled to the adenylate cyclase system.

Ontogeny of the hyobranchial apparatus in the salamanders Ambystoma talpoideum (Ambystomatidae) and Notophthalmus viridescens (Salamandridae): The ecological morphology of two neotenic strategies.

Comparison of metamorphosis of skull and hyobranchial system in two species of neotenic salamanders reveals two different types of neoteny. Ambystoma talpoideum is completely neotenic owing to delayed metamorphosis. Notophthalmus viridescens exhibits limited neoteny as a result of incomplete metamorphosis. Morphological details of neoteny are compared to life history in both species in order to discuss the ecological morphology of the two neotenic strategies. Comparisons to Taricha granulosa, Triturus vulgaris, and Ambystoma gracile indicate that these two strategies are widely employed and may represent familial patterns.

Adrenoceptor types in the respiratory vasculature of the salamander gill.

Adrenoreceptor types were determined in the branchial respiratory vasculature of the neotenic tiger salamander, Ambystoma tigrinum. Phenoxybenzamine antagonized increases in branchial vascular resistance caused by both epinephrine and norepinephrine. Propranolol antagonized both epinephrine- and norepinephrine-induced dilation of this vascular bed. Isoproterenol produced solely vasodilation, phenylephrine had no effect, and methoxamine caused constriction only at a very high dose. It is concluded that alpha-adrenoceptors mediate the catecholamine-induced vasoconstriction, and beta-adrenoceptors the catecholamine-induced vasodilation in the respiratory circulation of the gill.

Diurnal and seasonal variations in thyroid function of neotenic tiger salamanders (Ambystoma tigrinum).

No dirunal variations for plasma thyroxine (T4) in neotenic tiger salamander larvae were apparent in October, and T4 levels at 1200, 1800, and 2400 hr exceed those observed in May. A significant response to ovine thyrotropin (TSH) occurred only at 0600 hr in October. Plasma T4 and triiodothyronine (T3) were greatest in May at 0600 and 1800 hr, respectively, and ovine TSH significantly elevated T3 and T4 levels at all times except for T4 at 0600 when the maximal T4 level was observed. Maximal endogenous T4 (May) and maximal sensitivity to TSH (October) occurred at the end of the scotophase when plasma melatonin levels were greatest. Circulating levels of melatonin appear to be positively associated with thyroid function in this species.

Isolated perfused salamander proximal tubule: methods, electrophysiology, and transport.

Techniques are presented for the isolation and perfusion of renal proximal tubules from the neotenic salamander Ambystoma tigrinum. Methods are described for a determination of normal values for fluid transport and electrophysiological parameters. Stable cellular microelectrode recordings are reported that constitute the first intracellular measurements in an isolated perfused tubule preparation. With identical solutions in lumen and bath, fluid reabsorption averaged 0.28 nl.min-1.mm-1, transepithelial potential difference averaged -4.5 mV, transepithelial resistance was 52.1 omega.cm2, and the transepithelial chloride-to-sodium transference number ratio was 3.4. The basolateral cell membrane potential difference averaged -59.6 mV, and the ratio of apical-to-basolateral cell membrane resistance was between 3.9 and 5. Viability of the isolated perfused salamander proximal tubule preparation is demonstrated by a detailed comparison of the present data with results of in vivo micropuncture experiments on both Necturus and intact Ambystoma kidneys. In addition to being an advantageous preparation for long-term intracellular recordings, the Ambystoma kidney is unique in that proximal tubules can be studied both in isolation and by conventional micropuncture.

Vitellogenin synthesis and characterisation of the liver estrogen receptor in the neotenous salamander Ambystoma mexicanum

The neotenous salamander Ambystoma mexicanum reaches sexual maturity without completing metamorphosis. Females must, therefore, synthesize vitellogenin, the precursor of the egg-yolk proteins. We show that livers of female axolotls synthesize and secrete a phosphoprotein which migrates with Xenopus vitellogenin on SDS-gels and is precipitated by antibody prepared against Xenopus vitellogenin. The livers of male axolotls do not normally synthesize this protein but can be induced to do so by treatment in vivo with estradiol. A receptor with a high affinity for estradiol ( K d = 0.3 × 10 −9 M) was found in the nuclei prepared from livers of male and female axolotls. It sediments at 3.7 S at 0°C in sucrose gradients containing 0.5 M KCl. Each nucleus contains about 1300 binding sites for estradiol, 13 times the number found in normal male Xenopus nuclei, but as axolotl nuclei are about 12 times larger, the concentrations of binding sites are similar. In contrast to Xenopus, there is no detectable increase in the number of nuclear binding sites following estrogen treatment. We conclude that the controls affecting both the appearance of vitellogenin inducibility and the induction of vitellogenin synthesis differ between the two species A. mexicanum and Xenopus laevis.

Evidence for a highly differentiated sex chromosome heteromorphism in the salamander Necturus maculosus (Rafinesque)

The mitotic chromosomes of the neotenic (sensu Gould, 1977, and Alberch et al., 1979) salamander Necturus maculosus (Rafinesque) have been examined using a C-band technique to demonstrate the distribution of heterochromatin. The C-banded mitotic chromosomes provide evidence of a highly differentiated XY male/XX female sex chromosome heteromorphism, in which the X and Y chromosomes differ greatly in size and morphology, and in the amount and distribution of C-band heterochromatin. The X chromosome represents one of the largest biarmed chromosomes in the karyotype and is indistinguishable from similar sized autosomes on the basis of C-band heterochromatin. The Y chromosome, on the other hand, is diminutive, morphologically distinct from all other chromosomes of the karyotype, and is composed almost entirely of C-band heterochromatin. The discovery of an X/Y chromosome heteromorphism in this species is consistent with the observation by King (1912) of a heteromorphic spermatogenic bivalent. Karyological and phylogenetic implications are discussed.

A bibliography of larval and neotenic salamander biology

A bibliography of larval and neotenic salamander biology

Thyroxine-Induced Activation of Hypothalamo-Hypophysial Axis in Neotenic Salamander Larvae

Thyroxine injected into the hypothalamus of neotenic Ambystoma tigrinum induces metamorphosis by activating hypothalamo-hypophysial stimulatory control of thyroid activity, thereby removing the hypothalamic block to metamorphosis.

Concentrations of lactic acid in neotenic and transformed tiger salamanders (Ambystoma tigrinum) before and after activity

Concentrations of lactic acid in bodies of inactive neotenic tiger salamanders were similar to those recorded for inactive transformed animals. Transformed salamanders swimming against a water current for 5 min accumulated significantly more lactate in their body fluids than did neotenic salamanders, but subsequently metabolized the lactate at higher rates than characterized neotenic animals. Total body lactate in both morphs decreased rapidly in the first 1–1/2 h after activity, and within 6 h had returned to the level (common to both morphs) observed prior to activity. Whereas neotenic animals apparently swim with greater efficiency than transformed salamanders (4.26 μmole ATP/gvs. 6.06 μmole ATP/g expended by neotenic and transformed animals, respectively, during 5 min of continuous swimming), the transformed animals exhibit a metabolic adaptation to aquatic existence enabling them rapidly to recover from a lactacid debt.

The Life Cycle of Cephalogonimus salamandrus sp. n. (Digenea: Cephalogonimidae) from Ambystoma tigrinum (Green) from Eastern Washington

Mother and daughter sporocysts and xiphidiocercariae develop in Helisoma trivolvis. Cercariae penetrate tadpoles of Rana pretiosa and small A. tigrinutm larvae in which they encyst. The adult develops in the anterior small intestine of A. tigrilnum after ingestion of an infected 2nd intermediate host. The life cycle of C. salamandrus is compared with C. americanus from Rana clamitans. C. salamandrus is described as a new species on the basis of cercarial and adult morphology, and the failure to achieve cross-infections in the respective definitive hosts for C. americanus and C. salamandrus. Cephalogonimus Poirier, 1886, is the only genus of the family Cephalogonimidae in amphibians. Rai (1961) listed Cephalogonimus brevicirrus (Ingles, 1932), C. amphiumae (Chandler, 1923), C. letusus Dujardin, 1845, and C. am?ericanus (Stafford, 1902) from amphibians of North America. Premvati (1969) described C. sireni from Florida mud eels. The only life cycle reported is for C. americanus from Rana clamitans Latreille (Lang, 1968). This parasite has also been reported from R. catesbeiana Shaw (Rankin, 1945), and R. pipiens Schreber (Brandt, 1936; Najarian, 1955). An undescribed cephalogonimid is a common intestinal parasite of neotenic and adult Ambystoma tigrinum (Green) of eastern Washington. Of the other species of Cephalogonimus, this parasite most closely resembles C. americanus. Comparison of the life cycle stages of this parasite with those of C. americanus and the demonstrated specificity of these two parasites to their respective definitive hosts establishes the validity of this new species to which the name Cephalogonimus salamandrus is assigned. MATERIALS AND METHODS Eggs were teased from adult flukes and stored in filtered spring water at 5 C until used. Fully embryonated eggs were fed to young laboratoryreared Helisoma trivolvis (Say). Exposed snails were crushed and examined at intervals after Received for publication 9 April 1973. *A portion of this study was conducted at the University of Michigan Biological Station. tPresent address: Department of Biology, New Mexico State University, Las Cruces, New Mexico 88003. exposure. Naturally infected H. trivolvis were collected for cercarial study and laboratory infection of the second intermediate host. Cercariae were studied alive with and without vital stains. Cercariae were placed wvith various aquatic invertebrates, small salamander larvae, tadpoles, and small frogs. Penetration and encystment occurred only in amphibian skin. Metacercariae were studied at 2 hr, 1 day, 5 days, 10 days, 12 days, and 21 days after infection. Tadpoles and salamander larvae used as intermediate hosts were laboratory-reared. Large neotenic A. tigrinum for use in laboratory infections were collected from a lake where no trematode or cestode parasites were found in 150 A. tigrinum and no Helisoma spp. have been found in over 6 years of study. Salamanders were maintained in filtered spring wrater on a diet of earthworms. Before exposure 13 neotenic salamanders (20 to 30 cm in length) were starved for 2 weeks. Feeding was resumed the day after exposure. Each of 11 salamanders was permitted to ingest two infected tadpoles, each containing 20 to 30 metacercariae which were 12 days old at exposure. Salamanders were necropsied at 52 hr, 5 days, 10 days, 15 days, 20 days, and 48 days after exposure. Also each of two salamanders was permitted to ingest two infected salamander larvae, each containing 20 to 30 metacercariae which were 12 days old at exposure. These were necropsied at 5 days. Recovered flukes were studied alive, measured, heat-killed, stored in AFA, and stained in Semichon's Carmine. To determine if A. tigrinum and R. pretiosa of eastern Washington could be infected with C. americanus, hosts were transported to the University of Michigan Biological Station. Methods for handling C. americanus and collection of Cephalogonimus-free R. clamitans have been reported (Lang, 1968). Two salamanders were exposed to 10-day-old metacercariae of C. americanus. Each salamander received 50 to 70 metacercariae and was necropsied 10 or 30 days after exposure. During this experiment two A. tigrinum

Effects of mammalian gonadotropins (LH, FSH, HCG) and gonadal steroids on TSH-induced metamorphosis of Ambystoma tigrinum (Amphibia: Caudata)

The hypothesis that gonadotropins and/or gonadal steroids inhibit metamorphosis of neotenic salamanders was investigated in the tiger salamander, Ambystoma tigrinum. Sexually immature larvae were pretreated for 2 weeks with gonadotropins (LH and/or FSH, HCG) or gonadal steroids (testosterone and/or estradiol-17β) followed by continued treatment plus TSH. None of the hormones tested inhibited TSH-induced metamorphosis. LH, in fact, promoted metamorphosis during the pretreatment period, and testosterone accelerated TSH-induced metamorphosis. The induction of metamorphosis by LH can be accounted for in terms of TSH contamination. FSH did not contain sufficient TSH contamination to induce metamorphosis at the dosage employed. The importance of sensitivity of amphibian thyroid glands to mammalian TSH and the implications of TSH contaimination of pituitary gonadotropin preparations are discussed.

The Occurrence of Neotenic Salamanders, Ambystoma tigrinum diaboli Dunn, in Devils Lake, North Dakota

The Occurrence of Neotenic Salamanders, Ambystoma tigrinum diaboli Dunn, in Devils Lake, North Dakota

Ultrastructure of thyroid follicle cells of three salamanders ( Ambystoma, Amphiuma, and Necturus) exhibiting varying degrees of neoteny

Thyroid follicle cells of a facultively neotenic salamander ( Ambystoma tigrinum ), a perpetual perennibranchiate ( Necturus maculosus ), and a consistently partially metamorphosing derotreme ( Amphiuma means ), were compared at light and electron microscopic levels. In all three urodeles, a feltwork of cytoplasmic filaments apparently becomes contractile and is responsible for relatively rapid changes in cellular configuration. It apperars that microtubules form as an intermediate step in the synthesis of new filaments. The paucity of parafollicular (light) cells may be partly responsible for the lack of ossification in adult perennibranchiates. While recent evidence suggests that little T 4 is produced and none is released into the blood stream by Necturus thyroid ( 42 ), morphologically, the follicle cells appear to be relatively active. Detection of small amounts of T 4 in the thyroid of this genus may merely represent a disappearing function for this gland. Therefore, it might prove useful to look for other functions for the follicle cell of Necturus without the preconceived notion that it only synthesizes iodinated tyrosines.

An electron microscopic study of the choroid plexuses of Necturus maculosus.

AbstractThe choroid plexuses of the neotenous salamander, Necturus maculosus, were examined by light and electron microscopy. Their epithelium was found to consist of a single layer of flattened to cuboidal cells resting upon a well‐developed, continuous basement membrane. The cytoplasm of a typical epithelial cell contained, besides a large, centrally‐placed nucleus, numerous mitochondria, an extensive agranular endoplasinic reticulum, prominent Golgi complexes and a variety of dense bodies. In addition, occasional clusters of ribosomes, lipid droplets, fascicles of fine filaments and numerous glycogen granules were present within the cytoplasm. Specializations of the apical (ventricular) surfaces of the epithelial cells included an irregular brush border, cilia, and minute, possibly micropinocytotic, invaginations of the plasma membrane. Surfaces of contacts between epithelial cells were specialized by “tight junctions” desmosomes, and complex inter dictations of the apposed plasma membranes. The interstitial space separating the epithelial layer from the thin‐walled, non‐fenestrated plexus blood vessels contained a mesh‐work of collagen fibrils and a variety of connective tissue cell types. Electron dense Thorotrast particles were found to traverse the endothelial walls of the blood vessels and accumulate within the interstitial spaces within 30 minutes after being injected into the blood stream. The epithelial basement membrane, however, apparently prevented the tracer particles from reaching the epithelial cells, and the ventricular spaces beyond, as long as five hours after injection.

COMPARATIVE STUDIES OF THE OXYGEN CONSUMPTION OF THREE SPECIES OF NEOTENIC SALAMANDERS AS INFLUENCED BY TEMPERATURE, BODY SIZE, AND OXYGEN TENSION

1. Three species of neotenic salamanders of the genus Eurycca were collected from their specific, though not widely separated, habitats on the escarpment region of the Edwards plateau in south central Texas.2. Utilizing standard Warburg manometry, oxygen consumption was determined for each of the species (with and without shaking) at 15, 20, 25. and 30° C. Maximal respiratory rates were exhibited by Eurycea neotenes at 30° C., by Eurycea nana at 25° C., and by Eurycea pterophila at 20° C. Mechanical stimulation (shaking) increased the rate of oxygen uptake but did not alter the shape of the R/T curves. For E. nana and E. neotenes the temperature at which the maximal metabolic rates were observed appeared to be correlated with the temperature characteristics of the natural environment.3. There is a decrease in metabolic rate with increasing body size. The specific relationship is in good agreement with previously reported values.4. All three species of salamanders seem to be intermediate between oxygen conformers amid regulators, in that the rate of oxygen consumption is dependent on oxygen concentration (up to the oxygen content of air), but not in a strictly linear manner.