Reassessment of the environmental mechanisms controlling developmental polyphenism in spadefoot toad tadpoles

Identifying the environmental mechanism(s) controlling developmental polyphenism is the first step in gaining a mechanistic and evolutionary understanding of the factors responsible for its expression and evolution. Tadpoles of the spadefoot toad Spea multiplicata can display either a typical omnivorous or a carnivorous phenotype. Exogenous thyroxine and feeding on conspecific tadpoles have been accepted as triggers for development of the carnivorous phenotype on the basis of a series of studies in the early 1990s. I repeated the thyroxine and conspecific-feeding assays and demonstrated that neither exogenous thyroxine nor feeding on conspecifics induces the carnivorous phenotype. Previous researchers used simple ratio statistics to argue that field-collected carnivores and thyroxine-treated tadpoles are similar, and my results supported these claims if I used the same simple ratio methodology. However, investigation of trait developmental trajectories and allometries for field-collected carnivores and thyroxine-treated and conspecific-fed tadpoles show that these phenotypes are profoundly different.     

Emerging model systems in eco-evo-devo: the environmentally responsive spadefoot toad

Spadefoot toads have emerged as a model system for addressing fundamental questions in ecological and evolutionary developmental biology (eco-evo-devo). Their tadpoles produce a wide range of adaptive phenotypes in direct response to diverse environmental stimuli. Such phenotypic plasticity offers an excellent opportunity to examine how an organism's ecology affects its development as well as how an organism's development influences its ecology and evolution. By characterizing and understanding the interconnectedness between an organism's environment, its development responses, and its ecological interactions in natural populations, such research promises to clarify further the role of the environment in not only selecting among diverse phenotypes, but also creating such phenotypes in the first place.     

Morphological correlates of sprint swimming speed in five species of spadefoot toad tadpoles: Comparison of morphometric methods

The relationship between vertebrate morphology and swimming performance has long interested biologists. Recent work on predator‐induced morphological plasticity of anuran tadpoles has increased this interest. Here, I use data on five species of spadefoot toad tadpoles (Scaphiopodidae) to compare linear and geometric morphometrics. Linear measures explain only 7–26% of the variation in swimming speed, depending on species, whereas geometric morphometrics could explain 24–46% of the same variation. I also compare two methods for examining how similar the morphology–swimming speed relationship is among species. A canonical variate derived from a MANCOVA approach successfully detected species differences in these relationships, whether using linear or geometric methods, but a canonical correlation approach failed in both cases. Overall, tadpoles with smaller bodies, larger tails, and larger tail muscles are faster swimmers but the details of how these shape changes are achieved differed among species. For example, in some species a smaller body was achieved primarily by reducing abdomen size, whereas in others both the head and abdomen are smaller. Faster swimmers also had deeper tails, especially in the posterior half of the tail. This pattern would have been missed in standard linear morphometrics which usually only measures maximum tail depth. J. Morphol. 271:1044‐1052, 2010. © 2010 Wiley‐Liss, Inc.     

Metabolic reorganization and signal transduction during estivation in the spadefoot toad

The maximal activities of 28 enzymes, representing multiple pathways of intermediary metabolism, were quantified in the brain, liver and skeletal muscle of spadefoot toads Scaphiopus couchii, comparing control toads with animals that had estivated for 2 months. Estivation-induced changes in brain enzyme activities were consistent with suppressed glycolysis and increased ketone body and amino acid catabolism. In liver, estivation resulted in reduced activities of eight enzymes representing carbohydrate, amino acid, ketone body and phosphagen metabolism, but the maximal activity of malic enzyme increased by 2.4-fold. Estivation led to a large-scale reorganization of skeletal muscle affecting most of the enzymes analyzed. Activities of enzymes of carbohydrate catabolism were generally elevated except for glycogen phosphorylase and hexokinase, whereas those of enzymes of fatty acid synthesis and ketone body metabolism were reduced. Increased glutamate dehydrogenase activities in both brain and muscle, as well as activities of other amino-acid-catabolizing enzymes in muscle, correlated with specific changes in the free amino acids pools in those tissues (reduced glutamine activity, increased glutamate, alanine and valine activities) that appear to be related to protein catabolism, for the purposes of elevating urea levels. The effects of estivation on signal transduction systems were also assessed. Total activities of protein kinases A and C (PKA and PKC) were largely unaltered in toad tissues during estivation (except for a 57% reduction in liver total PKC), but in seven organs there were strong reductions in the percentage of PKA present as the active catalytic subunit in estivating animals, and three contained a much lower percentage of membrane-bound active PKC during estivation. Activities of protein phosphatase types 1, 2A, 2B, and 2C were also frequently reduced during estivation. Overall, these results suggest that anuran estivation involves metabolic reorganization, including changing the maximal activities of key enzymes of intermediary metabolism as well as depressing the metabolic rate by suppressing signal transducing enzymes.     

Evolutionary reduction of developmental plasticity in desert spadefoot toads

Organisms vary their rates of growth and development in response to environmental inputs. Such developmental plasticity may be adaptive and positively correlate with environmental heterogeneity. However, the evolution of developmental plasticity among closely related taxa is not well understood. To determine the evolutionary pattern of plasticity, we compared plasticity in time to and size at metamorphosis in response to water desiccation in tadpoles among spadefoot species that differ in breeding pond and larval period durations. Like most tadpoles, spadefoot tadpoles possess the remarkable ability to accelerate development in response to pond drying to avoid desiccation. Here, we hypothesize that desert spadefoot tadpoles have evolved reduced plasticity to avoid desiccation in ephemeral desert pools compared to their nondesert relatives that breed in long-duration ponds. We recorded time to and size at metamorphosis following experimental manipulation of water levels and found that desert-adapted species had much less plasticity in larval period and size at metamorphosis than nondesert species, which retain the hypothetical ancestral state of plasticity. Furthermore, we observed a correlation between degree of plasticity and fat body content that may provide mechanistic insights into the evolution of developmental plasticity in amphibians.     

The dynamics of developmental system drift in the gene network underlying wing polyphenism in ants: a mathematical model

SUMMARY Understanding the complex interaction between genotype and phenotype is a major challenge of Evolutionary Developmental Biology. One important facet of this complex interaction has been called "Developmental System Drift" (DSD). DSD occurs when a similar phenotype, which is homologous across a group of related species, is produced by different genes or gene expression patterns in each of these related species. We constructed a mathematical model to explore the developmental and evolutionary dynamics of DSD in the gene network underlying wing polyphenism in ants. Wing polyphenism in ants is the ability of an embryo to develop into a winged queen or a wingless worker in response to an environmental cue. Although wing polyphenism is homologous across all ants, the gene network that underlies wing polyphenism has evolved. In winged ant castes, our simulations reproduced the conserved gene expression patterns observed in the network that controls wing development in holometabolous insects. In wingless ant castes, we simulated the suppression of wings by interrupting (up- or downregulating) the expression of genes in the network. Our simulations uncovered the existence of four groups of genes that have similar effects on target gene expression and growth. Although each group is comprised of genes occupying different positions in the network, their interruption produces vestigial discs that are similar in size and shape. The implications of our results for understanding the origin, evolution, and dissociation of the gene network underlying wing polyphenism in ants are discussed.     

Retardation of thyroxine-induced metamorphosis by Amphenone B in toad tadpoles

The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.     

The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii)

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.     

The first find of a spadefoot toad (Anura,Pelobatidae) in the Miocene of Moldova

A skeleton fragment of a spadefoot toad (family Pelobatidae Bonaparte, 1850) from the Middle Miocene (Early Sarmatian) of the Karpov Yar locality (northern Moldova) is described. Since the material is incomplete, it is only determined as Pelobatidae gen. indet. This is the first spadefoot toad from the Miocene of Moldova.     

Automated acoustic monitoring of endangered common spadefoot toad populations reveals patterns of vocal activity

1. In the context of global amphibian decline, monitoring and restoration programmes are important. Acoustic monitoring is a possible approach for underwater vocalising species like the rapidly declining European common spadefoot toad (Pelobates fuscus). In this study, our aim was to design a dedicated software detector to be used in combination with programmable audio recorders to process the large amount of data generated by long-term acoustic monitoring and to use it for investigating the seasonal and circadian patterns of P. fuscus vocal activity.
2. The software detector targets advertisement calls of the species. Based on acoustic analysis of that call, we developed a detector that utilises both frequency and time features of the calls. Data collected during three breeding seasons in four known or potential P. fuscus breeding sites of north-eastern France were used to build a ground truth in order to test the performance of the detector. Then, we used the detector for analysing four acoustic monitoring campaigns conducted in two different sites over two breeding seasons to gain insight into the seasonal and circadian patterns of vocal activity of this species.
3. Evaluation of the P. fuscus call detector against a ground truth returned false-positive rates below 1.5% and true-positive rates ranging from 53% to 73%. These figures are compatible with long-term monitoring of the presence of the species. Running the software detector on standard hardware, the computation time for post-processing the 360 hr of a typical 3-month monitoring campaign was less than 1 day.
4. The seasonal pattern of P. fuscus underwater vocal activity is more complex than previously recognised. Over the whole ostensible 3-month breeding season, the actual time window for vocalising and breeding can last from a few days up to several weeks and may be split into clearly distinct episodes. When vocalisations occurred at both night- and daytime, the circadian vocal activity of P. fuscus occasionally proceeded uninterrupted for 24 hr but usually a several hour lull occurred immediately prior to sunset. When vocalisations occurred at both night- and daytime, the vocal activity pattern followed a bimodal distribution with a nocturnal highest peak of activity and a second peak occurring in the morning.
5. Our results demonstrate that it is feasible to monitor presence of P. fuscus in north-eastern France using a dedicated software detector combined with programmable audio recorders. Based on the outcomes of the detector applied to long-term audio data sets, we reveal temporal patterns of the vocal activity of the species and subsequently provide recommendations for attended and unattended acoustic monitoring.

     

Simulating viability of a spadefoot toad Pelobates fuscus metapopulation in a landscape fragmented by a road

A stochastic simulation model allowing for demographic and environmental stochasticity was developed in order to predict the dynamics of a Pelobates fuscus metapopulation. The metapopulation (consisting of ca 1000 adult individuals) was intensively studied in the field for a period of four years and the simulation model was parameterised (sex ratio, age‐specific survival rates, fecundity and dispersal between subpopulations) using field data. A sensitivity analysis revealed that a change in the juvenile yearly survival rate has a relatively larger effect on subpopulation persistence than adult survival rate and fecundity rate have. The probability of subpopulation persistence for one hundred years increased from 0 to 0.6 with a change in juvenile yearly survival rate from 0.35 to 0.40. Varying dispersal rate (from 0 to 1% of the individuals in a subpopulation moving to another subpopulation within a year) showed that four of the five subpopulations are dependent on the last one for persistence, indicating a source‐sink structure of the metapopulation. The subpopulation with the highest estimated juvenile survival has a far higher persistence probability than the others; they in turn would go extinct were it not for the occasional input of individuals from the source subpopulation. The source‐sink structure was also apparent when simulating the isolation effect of the road: persistence of the subpopulation isolated by the road decreases markedly with only a 20% decrease in the number of individuals dispersing to this pond. Environmental stochasticity decreases persistence time of the source subpopulation, but increases persistence time of the unstable ones. This is probably due to the presence of overcompensating density‐dependent factors affecting the subpopulations and to the more general effect of stochasticity: it may temporarily change reproduction and mortality rates, increase time to extinction for unstable subpopulations through a rescue effect; and decrease time to extinction for the more stable subpopulations.     

Ontogeny of corticotropin-releasing factor effects on locomotion and foraging in the Western spadefoot toad (Spea hammondii)

We investigated the effects of corticotropin-releasing factor (CRF) and corticosterone (CORT) on foraging and locomotion in Western spadefoot toad (Spea hammondii) tadpoles and juveniles to assess the behavioral functions of these hormones throughout development. We administered intracerebroventricular injections of ovine CRF or CRF receptor antagonist alphahelical CRF((9-41)) to tadpoles and juveniles, and observed behavior within 1.5 h after injection. In both premetamorphic (Gosner stage 33) and prometamorphic (Gosner stages 35-37) tadpoles, CRF injections increased locomotion and decreased foraging. Injections of alphahelical CRF((9-41)) reduced locomotion but did not affect foraging in premetamorphic tadpoles, but dramatically increased foraging in prometamorphic tadpoles compared to both placebo and uninjected controls. Similarly, alphahelical CRF((9-41)) injections stimulated food intake and prey-catching behavior in juveniles. These results suggest that in later-staged amphibians, endogenous CRF secretion modulates feeding by exerting a suppressive effect on appetite. By contrast to the inhibitory effect of CRF, 3-h exposure to CORT (500 nM added to the aquarium water) stimulated foraging in prometamorphic tadpoles. These tadpoles also exhibited a CORT-mediated increase in foraging 6 h after CRF injection, which was associated with elevated whole-body CORT content and blocked by glucocorticoid receptor (GR) antagonist (RU486) injections. Thus, exogenous CRF influences locomotion and foraging in both pre- and prometamorphic tadpoles, but endogenous CRF secretion in relatively unstressed animals does not affect foraging until prometamorphic stages. Furthermore, the opposing actions of CRF and CORT on foraging suggest that they are important regulators of energy balance and food intake in amphibians throughout development.     

Chemical defense of toad tadpoles under risk by four predator species

Many organisms use inducible defenses as protection against predators. In animals, inducible defenses may manifest as changes in behavior, morphology, physiology, or life history, and prey species can adjust their defensive responses based on the dangerousness of predators. Analogously, prey may also change the composition and quantity of defensive chemicals when they coexist with different predators, but such predator‐induced plasticity in chemical defenses remains elusive in vertebrates. In this study, we investigated whether tadpoles of the common toad (Bufo bufo) adjust their chemical defenses to predation risk in general and specifically to the presence of different predator species; furthermore, we assessed the adaptive value of the induced defense. We reared tadpoles in the presence or absence of one of four caged predator species in a mesocosm experiment, analyzed the composition and quantity of their bufadienolide toxins, and exposed them to free‐ranging predators. We found that toad tadpoles did not respond to predation risk by upregulating their bufadienolide synthesis. Fishes and newts consumed only a small percentage of toad tadpoles, suggesting that bufadienolides provided protection against vertebrate predators, irrespective of the rearing environment. Backswimmers consumed toad tadpoles regardless of treatment. Dragonfly larvae were the most voracious predators and consumed more predator‐naïve toad tadpoles than tadpoles raised in the presence of dragonfly cues. These results suggest that tadpoles in our experiment had high enough toxin levels for an effective defense against vertebrate predators even in the absence of predator cues. The lack of predator‐induced phenotypic plasticity in bufadienolide synthesis may be due to local adaptation for constantly high chemical defense against fishes in the study population and/or due to the high density of conspecifics.     

Postembryonic ontogeny of the spadefoot toad,Scaphiopus intermontanus (Anura: Pelobatidae): Skeletal morphology

Postembryonic skeletal ontogeny of the pelobatid frog Scaphiopus intermontanus is described based on a developmental series of cleared-and-stained, whole-mount specimens. The focus is on laboratory-reared individuals fed a herbivorous diet as larvae. Although there is variation in the timing of ossification of individual skeletal elements relative to developmental stages based on external morphological criteria, the sequence of skeletal development generally is conservative. Compared with its close relative, S. bombifrons, ossifications that occur during prometamorphosis tend to be slightly delayed in S. intermontanus; however, cranial bones that ossify during late metamorphic climax in S. intermontanus are delayed until postmetamorphosis in S. bombifrons. The differences in timing between the two species are consistent, however, with differences observed between two developmental series of S. intermontanus raised at two different temperatures. Noteworthy features of skeletal development in S. intermontanus include: 1) presence of palatine ossifications that form from independent centers of ossification and soon fuse with the postnarial portion of the vomers to form the compound vomeropalatine bones; 2) compound sphenethmoid that may arise from four or more endochondral centers of ossification and one dorsal, dermal center of ossification; and 3) presence of transverse processes and vestigal prezygapophyses on the first postsacral vertebra. The morphology of the larval orbitohyoideus and interhyoideus muscles is compared. The record of skeletal ontogeny and muscle morphology presented herein for the herbivorous larval morph can serve as a baseline for comparisons with the ontogeny of the carnivorous larval morph of Scaphiopus. J. Morphol. 238:179–244, 1998. © 1998 Wiley-Liss, Inc.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.     

宁陕齿突蟾蝌蚪的生物学特性

齿突蟾属(ScutigerTheobald,1868)包括齿突蟾亚属(ScutigerDubois,1980)和猫眼蟾亚属(AelurophryneFei,Ye and Li,1989),前者已知有10种,后者7种(费梁等,2005)。在我国有8种齿突蟾亚属物种分布于我国西南和青藏高原及周边地区,其中发现于陕西省宁陕县平河梁的宁陕齿突蟾(S·ningshanensis)是分布最东北的特有珍稀物种。尽管经过20多年来的多次采集,至今仅获得模式和配模标本,对其生物学特性了解甚少(方荣盛,1985;梁刚等,1989;费梁,1999),为这一珍稀齿突蟾的保护带来很大困难。蝌蚪是无尾两栖动物生命史中的一个重要时期,大约有四分之三…