Structure and dynamic of a natterjack toad metapopulation (Bufo calamita)

Summary The migratory and reproductive behaviour ofBufo calamita was studied at four neighbouring breeding sites in the northern Rhineland, Germany, from 1986 to 1991. Radio telemetry and marking systems based on toe-clipping and on microchips were used to follow the tracks of toads and for individual recognition. Emphasis lay on estimates of (1) the exchange of reproductive individuals between neighbouring sites, and (2) the reproductive success at each site. Allozyme electrophoresis served to assess the genetic diversity of local populations. More than 90% of all reproductive males showed a lifelong fidelity to the site of first breeding, whereas females did not prefer certain breeding sites. Due to the female-biased exchange of individuals among neighbouring sites the genetic distance between local populations was generally low but increased with geographical distance. This pattern of spatial relations is consistent with the structure of a metapopulation. Morever, up to three mass immigrations of males per breeding period, replacing previously reproductive individuals, suggested the existence of temporal populations successively reproducing at the same locality. Genetic distances were considerably greater between temporal populations than between local ones, indicating partial reproductive isolation. In fact, an exchange of reproductive individuals between the temporal populations at each site was not detected, but gene flow due to the recruitment of first-breeders originating from offspring other than their own seems probable. Thus, natterjack metapopulations consist of interacting local and temporal populations. The reproductive success differed considerably among the four sites and also between the temporal populations. Three out of four local populations had low reproductive success as well as the latest temporal population. The persistence of these populations depended entirely on the recruitment of juveniles from the only self-sustaining local population. This rescue-effect impeded local extinction. The discussion focuses on the modifications required to fit the classical metapopulation concept to the empirical findings and their consequences for the dynamics of amphibian metapopulations.     

Temporal spacing of breeding activity in the natterjack toad,Bufo calamita

Summary The reproductive behaviour of the toad Bufo calamita was studied in a large population in the northern Rhineland, Germany. Toads bred at four spatially separated but neighbouring areas including 34 breeding ponds. Three methods were applied to monitor quantitatively the intensity of breeding and migratory activity: capture-mark-recapture, a mechanical tracking device, and radio-telemetry. In 1987 a twofold temporal spacing of breeding activity was observed: (1) division of the reproductive period into early, main, and late breeding periods; (2) subdivision of each breeding period into several short calling periods. Since weather conditions were favourable during most of the reproductive period, environmental factors contributed little (23.4%–41.0%) to the temporal variation in breeding intensity as shown by multivariate statistics. The period of vocalization (April to August) included the activity of three distinct male groups. The first group called from April to mid-May (2 calling periods), the second from May to early July (5 calling periods) and the third from mid-July to August (2 calling periods). Within a calling period most males stayed near their calling sites and migratory activity was low. Extensive movements were detected when a new group of males immigrated from the feeding regions to the breeding areas and males that had been calling previously emigrated, at least partly. However, exchange of males among the neighbouring breeding choruses were clearly audible. It is suggested that the energetic costs of vocalization impeded continuous calling activity in individuals and accounted for the periodical variations in chorus size. The short-time variations of breeding activity therefore probably reflect the physiological limits of toads in a situation where the weather permits continuous reproductive activity.     

Ultrastructural changes in the spleen of the natterjack,Bufo calamita,after antigenic stimulation

Summary In the present study comparative aspects of the ultrastructure of the spleen were analyzed in non-immunized and T-dependent antigen-challenged natterjacks, Bufo calamita. Special attention is focused on the role of the non-lymphoid components in the splenic immunoreactivity. Ten days after primary immunization with sheep erythrocytes, splenic lymphoid follicles increase considerably in number and size. By that time, lymphoblasts, medium and large lymphocytes abound in the periphery of the white pulp near the marginal zone. Meanwhile, in the red pulp numerous monocytes migrating across the sinusoidal walls apparently transform into giant, dendritic-like cells. Twenty days after immunization the splenic lymphoid follicles decrease in number, although certain reactivity persists and numerous plasma cells occur in the cell cords and sinusoids of the red pulp. These results are discussed comparatively with those reported in other lower vertebrates.     

Microsatellite analysis of the natterjack toad (Bufo calamita) in Denmark: populations are islands in a fragmented landscape

The European natterjack toad (Bufo calamita) has declined rapidly in recent years, primarily due to loss of habitat, and in Denmark it is estimated that 50% of the isolated populations are lost each decade. To efficiently manage and conserve this species and its genetic diversity, knowledge of the genetic structure is crucial. Based on nine polymorphic microsatellite loci, the genetic diversity, genetic structure and gene flow were investigated at 12 sites representing 5–10% of the natterjack toad localities presently known in Denmark. The expected heterozygosity (H _E) within each locality was generally low (range: 0.18–0.43). Further analyses failed to significantly correlate genetic diversity with population size, degree of isolation and increasing northern latitude, indicating a more complex combination of factors in determining the present genetic profile. Genetic differentiation was high (overall θ = 0.29) and analyses based on a Bayesian clustering method revealed that the dataset constituted 11 genetic clusters, defining nearly all sampling sites as distinct populations. Contemporary gene flow among populations was undetectable in nearly all cases, and the failure to detect a pattern of isolation by distance within major regions supported this apparent lack of a gene flow continuum. Indications of a genetic bottleneck were found in three populations. The analyses suggest that the remaining Bufo calamita populations in Denmark are genetically isolated, and represent independent units in a highly fragmented gene pool. Future conservation management of this species is discussed in light of these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Density-related features of natterjack toad (Bufo calamita) population in Britain

We have studied a series of natterjack populations with varying densities of adults on the three main habitat types (lowland heaths, coastal dunes and coastal marshes) occupied by this species in Britain. Animals were aged by skeletochronology, condition was assessed by measuring weight length ratios in autumn and home ranges were determined by individual-marking and by radiotelemetry. Female longevity was inversely correlated with population density, but no similar effect was observed for males. Uniformity of growth rates, absolute growth rates and mean condition for both sexes were also inversely related to population density, whereas mean home range showed a positive correlation. Taken together the data indicated that in high-density populations natterjack toads grew more slowly and erratically, were in poorer condition and foraged much more widely than their counterparts in low-density populations.     

Phylogeography of the natterjack toad Bufo calamita in Britain: genetic differentiation of native and translocated populations

The natterjack toad Bufo calamita is rare in Britain, which is at the northwestern edge of its biogeographical range. We investigated the level of genetic differentiation amongst almost all (34 out of 38) of the surviving British populations of this species, and among six new populations established by translocations during the 1980s. For eight microsatellite loci, allele sizes and frequencies were analysed using samples from each of these populations. The populations clustered into three robustly differentiated groups, each of which corresponded with a geographical region (east/southeast England, Merseyside and Cumbria). The Cumbrian populations showed a further weak geographical substructuring into northern and southern clades. The populations in south Cumbria were genetically more diverse than those in any of the other regions, as judged by the mean numbers of alleles per locus and the mean heterozygosity estimates. The translocated populations clustered close to their founders and, with one exception, did not differ significantly with respect to mean allele numbers, heterozygosity or polymorphism level. However, significant genetic differentiation (as measured by unbiased R _ST) was found between all but one of the founder-translocation pairs. The implications of this phylogeographic study for the future conservation of B. calamita in Britain are discussed.     

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.     

Reconciling Genetic and Demographic Estimators of Effective Population Size in the Anuran Amphibian Bufo Calamita

We used genetic and demographic methods to estimate the variance effective population sizes (N _e) of three populations of natterjack toads Bufo calamita in Britain. This amphibian breeds in temporary pools where survival rates can vary among families. Census population sizes (N) were derived from spawn string counts. Point and coalescent-based maximum likelihood estimates of N _e based on microsatellite allele distributions were similar. N _e/N ratios based on genetic estimates of N _e ranged between 0.02 and 0.20. Mean demographic estimates of N _e were consistently higher (2.7–8.0-fold) than genetic estimates for all three populations when variance in breeding success was evaluated at the point where females no longer influence their progeny. However, discrepancies between genetic and demographic estimators could be removed by using a model that included extra variance in survivorship (above to Poisson expectations) among families. The implications of these results for the estimation of N _e in wild populations are discussed.     

Behavioural thermoregulation of the Andean toad (Bufo spinulosus) at high altitudes

Summary The body temperature of free-ranging Andean toadsBufo spinulosus was measured either directly or radiotelemetrically during two 15-day periods at 3200 m elevation in the Mantaro Valley, Central Perú. All toads attempted to maintain their diurnal sum of body temperature within a narrow range. Consequently thermoregulatory behaviour differed according to cloud cover and precipitation. If the sky was clear, toads emerged from their hiding place and exposed themselves to solar radiation during 3–5 h in the morning. Core temperature increased up to 15° C above the air temperature in shade and reached maximum values of about 32° C. At air temperatures (in sun) exceeding 29° C, toads maintained body temperatures below 32° C by evaporative cooling. Following heliothermic heating during the moring toads retreated to the shade, thereby decreasing body temperature below air temperature. Under overcast sky toads remained exposed during the whole day displaying body temperatures at or slightly above ambient levels. Quantitative models to predict the core temperature of toads under the different weather conditions demonstrated that the substrate temperature was the main energy source accounting for 64.6–77.9% of total variance whereas air temperature was of minor importance (1.5–4.4%). The unexplained variance was probably due to evaporative cooling. The volume of urine stored into the urinary bladder of toads varied diurnally; during basking in the morning hours most bladders contained large volumes of urine, whereas during the afternoon the bladders were mostly empty. The bladder contents probably serve as water reserves during basking when evaporative water loss was high. Toads preferred sites that provided shady hiding places as well as sun-exposed bare soil within a radius of 5 m. However, they frequently changed their centers of activity and moved to other sites in 20–70 m distance after periods of 2–5 days. The helio-and thigmothermic behaviour of the Andean toad permits the maintenance of high core temperature during morning which probably increases the digestion rate and accelerate growth. Evaporative cooling and preference of shady sites were employed to regulate body temperature below the morning levels in response to the constraints of water balance. Periodic changes between thigmothermic behaviour and locomotory activity during the night maintains body temperature above air temperature and prolongs the period of food uptake.Dedicated to Prof. Dr. H. Schneider on the occasion of his sixtieth birthday     

A comparison of single‐sample effective size estimators using empirical toad (Bufo calamita) population data: genetic compensation and population size‐genetic diversity correlations

The accuracy and precision of four single‐sample estimators of effective population size, N_e (heterozygote excess, linkage disequilibrium, Bayesian partial likelihood and sibship analysis) were compared using empirical data (microsatellite genotypes) from multiple natterjack toad (Bufo calamita) populations in Britain (n = 16) and elsewhere in Europe (n = 10). Census size data were available for the British populations. Because toads have overlapping generations, all of these methods estimated the number of effective breeders N_b rather than N_e. The heterozygote excess method only provided results, without confidence limits, for nine of the British populations. Linkage disequilibrium gave estimates for 10 British populations, but only six had finite confidence limits. The Bayesian and sibship methods both produced estimates with finite confidence limits for all the populations. Although the Bayesian method was the most precise, on most criteria (insensitivity to locus number, correlation with other effective and census size estimates and correlation with genetic diversity) the sibship method performed best. The results also provided evidence of genetic compensation in natterjack toads, and highlighted how the relationship between effective size and genetic diversity can vary as a function of geographical scale.     

Quantifying functional connectivity: experimental assessment of boundary permeability for the natterjack toad (Bufo calamita)

Like other pond-breeding amphibians, the natterjack toad (Bufo calamita) typically presents a patchy distribution. Because the species experiences high probabilities of local population extinction, its persistence within landscapes relies on both local and landscape-scale processes [dispersal allowing the (re)colonization of habitat patches]. However, the structure and composition of the matrix surrounding local populations can alter the dispersal rates between populations. As shown previously (Landscape Ecol 19:829–842, 2004), the locomotor performances of individuals at the dispersal stage depend on the nature of the component crossed: some landscape components offer high resistance to movement (high resistance or high viscosity components) whereas others allow high efficiency of movement (low resistance components). We now examine the ability of individuals to discriminate between landscape components and select low-resistance components. Our experimental study investigates the ways in which young natterjack toads choose from among landscape components common to southern Belgium. Toadlets (the dispersal stage) were experimentally confronted with boundaries between surrogates of sandy soils, roads, forests, agricultural fields and intensive pastures. Our results show: 1 the ability of toadlets to react to boundaries between landscape components, 2 differences in permeability among boundaries, and 3 our inability to predict correctly the permeability of the boundaries from the patch-specific resistance assessed previously. Toadlets showed a preference for bare environments and forests, whereas they avoided the use of agricultural environments. This pattern could not be explained in terms of patch-specific resistance only, and is discussed in terms of mortality risks and resource availability in the various landscape components, with particular attention to repercussions on conservation strategies.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

The evolution of offspring size and number: a test of the Smith-Fretwell model in three species of crickets

Most models of parental investment in offspring assume a trade-off between propagule size and number, and an increasing concave down function relating offspring fitness to propagule size. In this study, we test these two fundamental assumptions, using three closely related species of crickets, Gryllus firmus, G. veletis, and G. pennsylvanicus. Egg weight, 35-day fecundity and 35-day egg biomass were estimated in a population of each species, and the relationships between these reproductive traits and date of egg laying and body size were estimated. The relationships between egg weight and offspring survival were also sought for eggs buried at different depths, soil moistures, and soil types (G. firmus and G. veletis), as well as in the field (G. pennsylvanicus). A trade-off between egg weight and 35-day fecundity was revealed in a multivariate analysis taking into account among-species variation in egg weight and body size. Independent of the environmental conditions affecting the eggs, a positive correlation existed between the number of larvae that emerged from the soil and propagule weight in each species. Therefore, these findings provide partial support for the assumptions considered in the models mentioned above. A single optimal egg size was favored in two out of the three sets of conditions in which the functions relating egg weight to larval survival could be derived. The conditions encountered by the eggs, however, influenced the average survival of the larvae, as well as the shape of the relationship between egg weight and offspring survival. This suggests that cricket eggs frequently face heterogeneous environments with respect to egg and hatchling survival; the implication of habitat heterogeneity on the evolution of an optimal egg size is considered. The relationships between the reproductive components and female age and size, as well as between egg size and variation in cricket life-history, are discussed in an ecological and evolutionary context.     

Defining population boundaries: use of three Bayesian approaches with microsatellite data from British natterjack toads (Bufo calamita)

Defining boundaries between populations is often difficult in the absence of information about current levels of gene flow. Such definitions can be important, however, both for the understanding of population dynamics and for conservation planning. Recently developed Bayesian methods for analysing genetic data now provide a powerful approach to this problem. Natterjack toads Bufo calamita are endangered in Britain, where their distribution is restricted to four geographically discrete regions. In three of these regions the boundaries between populations are often uncertain. We therefore used Bayesian approaches with microsatellite data to try and define British natterjack population structure, and thus inform conservation management. A large sample of natterjack toads from all 38 locations in Britain where the species is native was genotyped at eight microsatellite loci. The genetic diversity of natterjack populations declined as a function of increasing latitude, echoing postglacial colonization dynamics. Comparisons of three assignment methods (structure, baps and geneland) generated some broad similarities but also some inconsistencies in the definitions of population structure, especially in the most complex region (south Cumbria). Implications of the analyses for the future conservation of Bufo calamita in Britain are discussed.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum)

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.     

Organogenesis and differentiation of the pancreas in the toad Bufo bufo L.

Summary In Bufo bufo at stage III₆ the first endocrine islets appear in the part of the pancreas corresponding to the dorsal anlage. At stage IV₂, 5 days later, the pancreatic duct develops and new islets arise by budding off from the ductal epithelium. The ultrastructural study of the secretory granules morphology of endocrine cells has distinguished four different cell types: B-cells (stage III₉), A-cells (stage IV₃), D-cells (stage IV₃) and a fourth type not yet identified (stage IV₃). By immunocytology insulin and corticotropin-releasing factor (CRF) cells have been demonstrated at stage III₉, and glucagon and somatostatin cells at stage IV₁. Lastly, endocrine islets can be homogeneous (predominantly containing insulin cells, rarely glucagon cells) or heterogeneous (insulin cells at the centre and glucagon or somatostatin cells at the periphery). Hypotheses are put forward for the origin and the constitution of the different generations of endocrine islets and isolated cells.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.