Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance and the restoration of functionally-effective behavior.     

Kin preference behaviour is present after metamorphosis in Rana cascadae frogs

Kin recognition was investigated in newly metamorphosed Rana cascadae frogs. Previous work has shown that larvae of this species prefer to associated with siblings over non-siblings. Juvenile frogs from three clutches were reared with siblings and tested for sibling preference as larvae and at 4–12 days and 39–47 days after metamorphosis. Tadpoles and froglets of the three clutches displayed a significant preference to associate with siblings.     

Differentiation of insect sensory neurons in the absence of their normal synaptic targets.

Sensory neurons in the antenna of the moth, Manduca sexta, arise and differentiate during the 18 days of metamorphosis from pupa to adult, sending axons to the brain. To assess the trophic dependence of developing antennal neurons on their targets, we studied antennae from surgically debrained animals. If the brain is removed 1 to 45 hr after pupal ecdysis (before and during the period when antennal neurons arise by cell divisions), adult development can be triggered by injection of β-ecdysone; if the brain is removed 50 to 60 hr after pupal ecdysis (before antennal axons contact the brain), metamorphosis proceeds spontaneously. Neurons proliferate normally and differentiate extensively in the antennae of debrained animals. They acquire a characteristic size and shape, elaborate axons and dendrites, migrate to appropriate positions in the sensilla, accumulate components of a neurotransmitter system (acetylcholine, choline acetyltransferase, and acetylcholinesterase), and generate electrical responses to olfactory and mechanical stimuli. Antennal sensory neurons thus differ from a variety of vertebrate neurons, which fail to mature when deprived of their synaptic targets.     

Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals

Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenopus laevis larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.     

Laboratory culture of the Nudibranch Tritonia diomedea bergh (Tritoniidae: Opisthobranchia) and some aspects of its behavioral development

Tritonia diomedea Bergh was reared from oviposition, through metamorphosis to reproductive maturity in the laboratory. The larvae of T. diomedea are planktotrophic and undergo considerable shell growth (from 144.6–329 μ average maximum shell length). Metamorphosis does not require induction, but there may be a preference to metamorphose in the presence of the probable adult prey, a small Virgularia sp. Larvae in cultures fed no food, Dunaliella tertiolecta Butcher Isochrysis galbana Parke, or Monochrysis lutheri Droop did not achieve metamorphic competence at near ambient sea-water temperatures (11.9±1.3 and 13.0±0.8°C). Larvae from cultures fed Monochrysis at room temperature (20.8±1.5°C) or fed a 1 : 1 mixture of Isochrysis and Monochrysis at near ambient sea-water temperatures did metamorphose. Even so, only those larvae fed the 1 : 1 mixture survived more than a few days following metamorphosis. Adult behavioral patterns developed gradually, feeding being first observed at 5 days, swimming in response to NaCl crystals at about 60 days, copulation at about 272 days, and oviposition at about 277 days after metamorphosis. Growth rates were determined for field collected Tritonia diomedea; smaller animals gained and lost weight relatively faster than larger animals.     

Short‐term larval food stress and associated compensatory growth reduce adult immune function in a damselfly

Abstract 1. In animals with a complex life cycle, larval stressors may carry over to the adult stage. Carry‐over effects not mediated through age and size at metamorphosis have rarely been studied. The present study focuses on the poorly documented immune costs of short‐term food stress both in the larval stage and after metamorphosis in the adult stage. 2. The present study quantified immune function [number of haemocytes, activity of prophenoloxidase (proPO) and phenoloxidase (PO)] in an experiment where larvae of the damselfly Lestes viridis were exposed to a transient starvation period. 3. Directly after starvation, immune variables were reduced in starved larvae. Levels of proPO and PO remained low after starvation, even after metamorphosis. In contrast, haemocyte numbers were fully compensated by the end of the larval stage, yet were lower in previously starved animals after metamorphosis. This can be explained as a cost of the observed compensatory growth after starvation. Focusing only on potential costs of larval stressors within the larval stage may therefore be misleading. 4. The here‐identified immunological cost in the adult stage of larval short‐term food stress and associated compensatory growth strongly indicates that physiological costs may explain hidden carry‐over effects bridging metamorphosis. This adds to the increasing awareness that the larval and adult stages in animals with a complex life cycle should be jointly studied, as trade‐offs may span metamorphosis.     

The effect of a juvenile hormone analogue on ecdysteroid titre during development and HnRNA formation in the moth,Ephestia cautella

The juvenile hormone analogue, methoprene was found to interfere with normal development of Ephestia in a manner dependent on age. Young embryos, prior to the stage of blastokinesis, and animals, shortly before and after pupation, were found to be the most sensitive to the compound. The JHA inhibited metamorphosis and produced giant larvae when it was given to immature larvae, however, when it was given to larvae 2–3 days prior to pupation or to young pupae it did not affect metamorphosis but prevented adult emergence. Comparison of the ecdysteroid titre determined in control and treated animals in the various developmental stages showed that JHA depressed the ecdysteroid titre totally only when it was given to young larvae and partially when it was applied shortly before pupation. It seems that the action of methoprene on ecdysone regulated systems and/or ecdysone producing systems in Ephestia appears to be mainly during the larval stage prior to the appearance of the small ecdysteroid peak and the formation of HnRNA in the transition period from larvae to pupae.     

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.     

Stimulation of terrestrial-substrate preferences and locomotor activity in newly transformed tiger salamanders (Ambystoma tigrinum) by exogenous or endogenous thyroxine

Recently transformed adult tiger salamanders (Ambystoma tigrinum) that had never resided on land were treated with increasing, alternate-day injections of thyroxine (experiment 1) or single injections of particular dosages of this hormone (experiment 2). In all cases control salamanders received equivalent volumes of saline on a similar schedule. In both experiments, subjects treated with thyroxine displayed significant levels of terrestrial-substrate preference in a moisture-gradient apparatus. Salinetreated controls never emerged from the water. In experiment 2, animals that received single thyroxine treatments also displayed heightened levels of land-directed locomotor activity. In experiment 3, blood plasma was collected from salamanders displaying spontaneous land preference and others displaying water preference. Radioimmunoassay for thyroxine indicated that salamanders that had moved to, and resided upon, a terrestrial substrate some time subsequent to metamorphosis had significantly higher plasma levels of this hormone than did animals preferring to remain submerged. These results indicate (1) that thyroxine stimulates, and possibly maintains, emergence from water and terrestrial preference in newly transformed tiger salamanders and (2) that this hormone stimulates a locomotor activity component associated with the movement of formerly aquatic adults to the terrestrial habitat.     

The genetic covariance between life cycle stages separated by metamorphosis

Metamorphosis is common in animals, yet the genetic associations between life cycle stages are poorly understood. Given the radical changes that occur at metamorphosis, selection may differ before and after metamorphosis, and the extent that genetic associations between pre- and post-metamorphic traits constrain evolutionary change is a subject of considerable interest. In some instances, metamorphosis may allow the genetic decoupling of life cycle stages, whereas in others, metamorphosis could allow complementary responses to selection across the life cycle. Using a diallel breeding design, we measured viability at four ontogenetic stages (embryo, larval, juvenile and adult viability), in the ascidian Ciona intestinalis and examined the orientation of additive genetic variation with respect to the metamorphic boundary. We found support for one eigenvector of G (g_obsmax), which contrasted larval viability against embryo viability and juvenile viability. Target matrix rotation confirmed that while g_obsmax shows genetic associations can extend beyond metamorphosis, there is still considerable scope for decoupled phenotypic evolution. Therefore, although genetic associations across metamorphosis could limit that range of phenotypes that are attainable, traits on either side of the metamorphic boundary are capable of some independent evolutionary change in response to the divergent conditions encountered during each life cycle stage.     

Effect of excretory-secretory products of some fouling organisms on settling and metamorphosis of the larvae of <Emphasis Type="Italic">Styela rustica</Emphasis> (Ascidiae)

The effect of the excretory-secretory products of some fouling animals on the settling and metamorphosis of larvae of the solitary ascidian Styela rustica was assessed. The substances secreted by the sponge Halichondria panicea stimulated settling of larvae, but concurrently blocked their metamorphosis. The excretory-secretory products of the mussel Mytilus edulis and the ascidian Molgula citrine did not affect settling of the S. rustica larvae but impeded their subsequent development. Water conditioned by the bivalve Hiatella arctica, stimulated settling and, apparently, metamorphosis of the larvae of S. rustica. The chemical substances produced by adult individuals of S. rustica facilitated settling of conspecific larvae but slightly delayed their metamorphosis.     

Facultative planktotrophy in the tropical echinoid Clypeaster rosaceus (Linnaeus) and a comparison with obligate planktotrophy in Clypeaster subdepressus (Gray) (Clypeasteroida: Echinoidea)

Larval development to metamorphosis and early juvenile growth and survivorship were examined in Clypeaster subdepressus (Gray) and C. rosaceus (Linnaeus). C. subdepressus has an obligatorily planktotrophic larva that metamorphoses after 16 to 28 days at 27°C. The larva of C. rosaceus can, but need not feed prior to metamorphosis, which occurs after 5 to 7 days at 27°C. Feeding by larvae of C. rosaceus does not change the time to metamorphosis but does increase size at metamorphosis, early juvenile growth and may increase juvenile survivorship relative to unfed larvae. Size at metamorphosis increases in larvae of C. rosaceus that feed for several days after they are competent to metamorphose, but there may be a limit to this increase because the condition of the rudiment degenerates after a period of time. The development of C. rosaceus may represent a transition between planktotrophy and lecithotrophy. This intermediate state has advantages for the juvenile stage that are not included in the trade of fecundity against risk to offspring usually considered in life history discussions of developmental mode of marine invertebrates.     

The effect of exposure to black background and to α-MSH on black-white background preference in the amphibian Xenopus laevis (Daudin)

Young frogs, Xenopus laevis, raised on a white background showed a slight preference for the black background of a black-white preference testing apparatus. Keeping frogs for either 24 hr or 20 days on a black background resulted in a further increase in their preference for black. Injecting 1 μg of synthetic α-MSH into white-adapted frogs had no effect on their black-white preference. On the other hand, Xenopus laevis tadpoles, at an early stage of metamorphosis, raised on a white background showed a significant preference for white. Keeping these tadpoles for 24 hr on a black background resulted in a shift from white to black preference. Injecting synthetic α-MSH into white-adapted tadpoles resulted in an increase in their preference for black. A behavioral feedback mechanism appears to exist between the release of endogenous MSH when a tadpole is exposed to a black background and its subsequent preference for background color.     

Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs⁺ or tumor-promoting phorbol esters (W. A. Müller (1985) Differentiation 29, 216–222). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [³H]-inositol. Upon exposure of the larvae to Cs⁺ the label in inositol trisphosphate (InsP₃) increased twofold as early as 15 sec after addition of Cs⁺. Within the first 60 sec the levels of inositol monophosphate (InsP₁) and inositol bisphosphate (InsP₂) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs⁺, only the label in InsP₂ was increased. When applied to saponin-permeabilized larvae, InsP₃ did not induce metamorphosis. But 1,2-dioctanoyl-rac-glycerol (diC₈) was effective in inducing metamorphosis with a half-maximal effective concentration of 9 μM. The percentage of metamorphosed animals after the application of 5 μM diC₈ (30 mM Cs⁺) was increased by the simultaneous application of 1 μM (0.1 μM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata.     

Skin toxin production of toads changes during early ontogeny but is not adjusted to the microbiota of the aquatic environment

Many animals display spectacular adjustments of their phenotype to changing environmental conditions in terms of altered behaviour, physiology and morphology, but whether similar plasticity in chemical defences is taxonomically widespread as well has remained unknown. Bufadienolides, toxins produced by some anuran amphibians, are known to be effective against many predators, but they can also contribute to defences against microorganisms. We experimentally investigated if qualitative and/or quantitative changes in the chemical defences of Bufo bufo tadpoles are induced by differences in the microbiota of their environment. We reared animals in purified tap water, or in pond water containing lowered or natural quantities of the natural microbiota, and measured the number of compounds and total quantity of bufadienolides at three ontogenetic stages during larval development, as well as 10 days after metamorphosis. We observed a significant decrease in the number of compounds, and a parallel increase in the total quantity of bufadienolides after metamorphosis, independent from treatments. The quantity of individual bufadienolide compounds also changed during ontogeny independently from treatments. Our results, thus, suggest that although bufadienolide production changes during early ontogeny, it is not adjusted to the microbiota present in the environment.     

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant,an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Effects of GABA and epinephrine on the settlement and metamorphosis of the larvae of four species of bivalve molluscs

Competent larvae of different marine bivalve species were treated with GABA and epinephrine at different concentrations and times of exposure to test the ability of the drugs to induce settlement and metamorphosis. GABA induced both settlement and metamorphosis in the mussel Mytilus galloprovincialis, the clams Venerupis pullastra and Ruditapes philippinarum and the oyster Ostrea edulis. Maximum induction of settlement (>39%) was achieved after exposure of V. pullastra larvae to 10⁻⁴ M GABA; this concentration of GABA also induced the highest percentages of metamorphosis in the four species studied. Epinephrine was identified as an active inducer of settlement and metamorphosis in bivalve molluscs. Exposure to 10⁻⁵ M epinephrine induced significant levels of settlement in Mytilus, Venerupis and Ostrea. In contrast, epinephrine failed to induce settlement behaviour in Ruditapes. Maximum induction of metamorphosis was produced by 10⁻⁵ M epinephrine in mussels, clams and oysters; Ruditapes showed the highest percentage of metamorphosis (>78%). This is the first report in which the involvement of GABA in the settlement and metamorphosis of bivalve molluscan larvae is demonstrated. It was also recognised that epinephrine plays a role not only in inducing metamorphosis but also in initiating settlement.     

Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

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.