The ontogeny of kin recognition in tadpoles of the toadBufo melanostictus (Anura; Bufonidae)

The ontogeny of kin recognition and influence of social environment on the development of kin recognition behaviour was experimentally investigated in tadpoles of Bufo melanostictus that lived in aggregations and showed low larval dispersion. Embryos and tadpoles of the toad were reared as (i) kin only, (ii) with kin and non-kin (separated by a mesh screen), and (iii) in isolation. They were tested for the ability to discriminate between (i) familiar siblings and unfamiliar non-siblings, (ii) familiar siblings and familiar non-siblings and, (iii) unfamiliar siblings and unfamiliar non-siblings. All tadpoles were fed on boiled spinach before conducting trials. Preference of test tadpoles to associate near the end compartments whether empty or containing members of specific stimulus groups was assessed using a rectangular choice tank. When tested in tanks with empty end compartments, the test tadpoles showed random distribution and thus no bias for the apparatus or the procedure. In the presence of kin/non-kin in the end compartments a significantly greater number of test tadpoles spent the majority of the time near familiar or unfamiliar kin rather than near familiar or unfamiliar non-kin. Kin discrimination ability persisted throughout larval development. Familiarity with siblings is not required for discriminating kin from non-kin, and kin discrimination ability is not modified following exposure to non-kin. Also, involvement of dietary cues is unlikely to be the prime mechanism of kin recognition inB. melanostictus unlike in some other anurans.     

水热环境对黑眶蟾蜍幼体存活的影响

禁食后在9种水热条件下,观测了不同温湿度对黑眶蟾蜍(Bufo melanostictus Schneider)幼体存活时间和动用体内贮能情况的影响。黑眶蟾蜍幼体在24℃浅水环境中存活时间长达22d。在高温和干燥条件下幼体的存活时间最短,32℃干燥环境中幼体存活时间不到1d。不同温湿度环境下幼体体内含脂量有显著差异。在高温和干燥环境中幼体存活时间短,动用贮能少,其体内脂肪含量和总能量动用少于其它温湿度环境中的幼体。结果表明,黑眶蟾蜍幼体适宜生活在24℃浅水和湿润环境中,觅食不足并非影响其幼体存活时间的主要因子,而高温和干燥是影响其幼体存活时间的关键因子。     

Visual performance of the toad (Bufo bufo) at low light levels: retinal ganglion cell responses and prey-catching accuracy

The accuracy of toad snapping towards moving worm dummies under various levels of dim illumination (from absolute threshold to moonlight) was videorecorded and related to spike responses of retinal ganglion cells exposed to equivalent stimuli. Some toads (at ca. 16 °C) successfully snapped at dummies that produced only one photoisomerization per 50 rods per second in the retina, in good agreement with thresholds of sensitive retinal ganglion cells. One factor underlying such high sensitivity is extensive temporal summation by the ganglion cells. This, however, is inevitably accompanied by very long response latencies (around 3 s near threshold), whereby the information reaching the brain shows the dummy in a position where it was several seconds earlier. Indeed, as the light was dimmed, snaps were displaced successively further to the rear of the dummy, finally missing it. The results in weak but clearly supra-threshold illumination indicate that snaps were aimed at the advancing head as seen by the brain, but landed further backwards in proportion to the retinal latency. Near absolute threshold, however, accuracy was too good, suggesting that the animal had recourse to a neural representation of the regularly moving dummies to correct for the slowness of vision.     

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus)

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e, the pituitary POMC cells, were not detected in any of the surgically treated animals while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.Abbreviations ACTH Adrenocorticotropic hormone - ANR anterior neural ridge - GH growth hormone - GTH gonadotropic hormone - MSH metanocyte stimulating hormone - NP central part of anterior neural plate - POMC proopiomelanocortin - PRL prolactin - TSH thyroid-stimulating hormone     

Capacitance,short-circuit current and osmotic water flow across different regions of the isolated toad skin

Summary The amphibian antidiuretic hormone, arginine vasotocin, stimulated osmotic water flow across isolated skin from the pelvic but not the pectoral skin of the toad, Bufo woodhouseii. Changes in the apical membrane capacitance were not observed for either region of the skin following treatment with arginine vasotocin when there was an osmotic gradient across the tissue. In the absence of an osmotic pressure gradient, the apical membrane capacitance of the pelvic skin increased from 2.8±0.5 to 3.3±0.6 F · cm^-2 after treatment with 5 · 10^-8 M arginine vasotocin. Under these conditions, apical membrane capacitance of the pectoral skin was 1.8±0.1 F · cm^-2 and did not change significantly after arginine vasotocin treatment. The amiloride-sensitive short-circuit current across the pelvic skin was stimulated by arginine vasotocin as was the density of channels in the apical membrane as determined by fluctuation analysis. Values for channel density in the pelvic skin also correlated with apical membrane capacitance and increased from 90 to 273 channels per m² of estimated membrane area following arginine vasotocin treatment. In the pectoral skin the stimulation of short-circuit current following arginine vasotocin treatment was small and an increase in channel density could not be demonstrated. The current through single Na⁺ channels in both regions of the skin did not different either before or after arginine vasotocin treatment.Abbreviations A amiloride - ADH antidiuretic hormone - AVT arginine vasotocin - C capacitance - C _a capacitance of apical membrane - f _c corner frequency - i single-channel current - osmotic water flow - IMP intramembrane particles - I _sc short-circuit current - amiloride-sensitive short-circuit current - M channel density - P _o probability of a channel being open - R channel receptor - R _a apical resistance - R _p paracellular resistance     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

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.     

Mitochondria-rich cells and carbonic anhydrase content of toad skin epithelium

Summary The density and carbonic anhydrase (CA) content of the mitochondria-rich cells (MRCs) in the skin epithelium of the toad, Bufo viridis, were studied under conditions of acclimation to various chlorinities. Long-term (days to weeks) acclimation to chloride-free solutions induced a great increase in the MRC density and the area occupied by the apical portion of these cells on the surface of the epithelium. The CA content of the epithelium, and individual MR cells, showed a 5- to 10-fold reduction after acclimation to solutions containing high chloride levels. The MRC density and their relative apical surface area correlated with the chloride permeability of the skin in acclimated (long-term) toads. It is concluded that the MRCs are the principal site of chloride permeability across the amphibian skin, and they respond in an adaptive manner to long-term changes in environmental chloride levels.This study was partially supported by the J. and A. Taub Fund for Biological Research at The Technion, and by the basic research fund of the Israel Academy of Sciences     

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     

Absence of the trade-off between the size and number of offspring in the natterjack toad (Bufo calamita)

Summary A trade-off between size and number of offspring was not found for females of similar sizes of the natterjack toad (Bufo calamita). Moreover, for large females, clutches with higher number of eggs had larger eggs as well. This suggests that larger females produce more numerous and larger eggs because they potentially have more energy available for reproduction. Egg size diminished allometrically with clutch size. Egg size, however, did not increase offspring fitness. Therefore, this allometric decrease may be considered a consequence of phylogenetic constraints rather than a result of optimizing selection.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

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.     

Histological effects of vitamin A on the tail amputated tadpoles ofBufo melanostictus

Vitamin A has manifold effects on the development, growth and pattern formation of several amphibians. At the same time it causes severe embryonic malformations. The histological changes brought about by vitamin A in the tail tissues of anurans are quite amazing. A common morphological change brought about by vitamin A in tail-amputated tadpoles ofBufo melanostictus include the formation of a large bulbular mass at the distal end of the tail following tail regeneration. Histology revealed that the bulbular mass consisted of notochordal cells only. Other histological changes are: a thickening of the epidermis and the basement membrane, enlargement of the notochord and the nerve cord, thickening of the sheath covering the notochord and the myelin sheath covering the nerve cord and the disorganization of muscle bundles. The significance of such changes is discussed.     

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.     

Membranes during yolk-platelet development in oocytes of the toad Bufo marinus

Summary Oocytes of the toad Bufo marinus have been studied by means of thin section and particularly freeze-fracture electron microscopy to characterize the cytoplasmic membranes around the yolk organelle, and the storage of yolk material in precursors and platelets. This appears to be a previously unknown type of yolk-platelet formation. During yolk-organelle development from the primordial precursor to the bi-partite fully grown yolk platelet, numerous lipoid droplets are attached to the periphery of the platelet, indicating an intense uptake of lipids. As is typical for amphibians, the fully grown yolk platelet has a crystalline internum covered by a dense osmiophilic externum, and the whole organelle is enveloped by a plasma membrane that shows no direct connection or fusion with endocytotic vesicles. The yolk membrane exhibits few intramembraneous particles (IMPs) at the core areas and some more where it borders fields of lipoid droplets. Here the IMPs show a net-like arrangement in the furrows between adjacent droplets.     

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.     

Expression pattern of glycoconjugates in the integument of Bufo ictericus (Anuran, Bufonidae): biochemical and histochemical (lectin) profiles

Mucous consists of glycoproteins and proteoglycans produced by specific secretory cells (mucocytes). In anurans the cutaneous mucous is produced by intradermal glands and displays both mechanical and chemical protection functions. Indeed, mucous maintains the integument moist and facilitates gas exchange (cutaneous respiration). In this work, the carbohydrate moiety distribution was investigated in the integument of Bufo ictericus using conventional and lectin histochemistry to describe the pattern of cutaneous glycoconjugate expression, including both secretory and structural proteoglycans. As a preliminary step, the descendent chromatography in Whatmann 1MM paper was undertaken to prepare the histochemical trials involving the lectins. In B. ictericus, the integument exhibits the basic morphological structure found in lower terrestrial vertebrates: the epidermis is a keratinized squamous stratified epithelium supported by spongious and compact layers. The spongy dermis contain secretory portion of both mucous and serous (or poison) glands. The paper chromatography identified galactose, fucose and mannose as characteristic sugar residues. The secretory cells of the mucous gland in the dermis, as well as the interstice between the stratum corneum and the subjacent stratum spinosum in the epidermis exhibit alpha-l-fucose and alpha-galactose residues. The serous glands give no reaction. The alpha-mannose residue was detected in the extracellular matrix of spongious dermis, but not in the dermal glands. The different glycoconjugate location reflects in two glycoconjugates categories: the secretory which participate in the water flow regulation, and the structural which is involved in the dermal maintenance.     

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI 1 and BMSI 2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI 1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 μM and 0.15 μM, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures.