Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea

Extracts prepared from the dried skins of approximately one hundred amphibian species from Australia and Papua New Guinea were subjected to biological screening in order to determine the nature and amounts of peptides active on smooth muscle preparations and systemic blood pressure present in these extracts. The most frequently and abundantly occurring peptides were those of the caerulein, bombesin and tachykinin peptide families represented, respectively, by caerulein; litorin, Glu(OMe)2-litorin and Glu(OEt)2-litorin; uperolein and Lys5-Thr6-physalaemin. Bradykinin-like peptides seem to have a rather diffuse distribution, in the species examined, but so far no peptide of this family has been isolated and sequenced. The only angiotensin-like peptide ever found in amphibian skin, crinia angiotensin II, has been isolated from skin extracts of a few species, belonging to the genera Crinia, Geocrinia, Ranidella and Litoria. The array of peptides occurring in amphibians from Australia and Papua New Guinea is destined to increase, because several apparently novel peptides have been identified in skin extracts by bioassay and radioimmunoassay.     

Antimicrobial peptides from the skins of North American frogs

North America is home to anuran species belonging to the families Bufonidae, Eleutherodactylidae, Hylidae, Leiopelmatidae, Ranidae, and Scaphiopodidae but antimicrobial peptides have been identified only in skin secretions and/or skin extracts of frogs belonging to the Leiopelmatidae (“tailed frogs”) and Ranidae (“true frogs”). Eight structurally-related cationic α-helical peptides with broad-spectrum antibacterial activity, termed ascaphins, have been isolated from specimens of Ascaphus truei (Leiopelmatidae) occupying a coastal range. Characterization of orthologous antimicrobial peptides from Ascaphus specimens occupying an inland range supports the proposal that this population should be regarded as a separate species A. montanus. Ascaphin-8 shows potential for development into a therapeutically valuable anti-infective agent. Peptides belonging to the brevinin-1, esculentin-1, esculentin-2, palustrin-1, palustrin-2, ranacyclin, ranatuerin-1, ranatuerin-2, and temporin families have been isolated from North American ranids. It is proposed that “ranalexins” represent brevinin-1 peptides that have undergone a four amino acid residue internal deletion. Current taxonomic recommendations divide North American frogs from the family Ranidae into two genera: Lithobates and Rana. Cladistic analysis based upon the amino acid sequences of the brevinin-1 peptides provides strong support for this assignment.     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.     

Terrestrial locomotion in two species of amphibian larva

Leptopelis hyloides and Chiromantis rufescens, which deposit eggs out of water, are compared with the aquatic breeding Bufo maculatus in terms of the ability of their larvae to move on land. Leptopelis larvae travelled further, faster and in a more consistent direction than Chiromantis larvae; they also survived better on land. Bufo larvae showed no terrestrial locomotion. These facts are related to the breeding behaviour of the three species. Locomotory ability in both Leptopelis and Chiromantis larvae was influenced by relative humidity and in Leptopelis the direction of movement was apparently influenced by topographic gradient and by the position of the closest standing water.     

Biogenic amines and active peptides in extracts of the skin of thirty-two European amphibian species

1. Extracts prepared from fresh or dried skins of 32 European amphibian species were submitted to chemical (colour reactions) and biological screening to determine the occurrence and contents of biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by tryptamine, 5-hydroxytryptamine, and its N-methylated, cyclized and sulphoconjugated derivatives. 3. The peptide families identified in the extracts were as follows: bombesins (bombesin and alytesin), bradykinins (bradykinin, bradykinin 1-8, bradykinin 1-7), chemotactic peptides (RECP I, II and III), bombinins and TRH. Bombesins, bombinins and TRH (thyrotropin-releasing hormone) were isolated from skin extracts of discoglossid frogs; chemotactic peptides and again TRH from extracts of ranid frogs. 4. Further research will certainly lengthen the list of active peptides in the skin of European amphibians, as is the case with Australian, American and African amphibians.     

Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than bufonids

1. Extracts prepared from dried or fresh skins of 52 African amphibian species, other than bufonids, were subjected to chemical (colour reactions) and biological screening, to determine occurrence and contents of aromatic biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by 5-hydroxytryptamine, its N-methylated derivatives and tryptamine. The indolealkylamines considered included the alkaloid trypargine, a carboline derivative resulting from the condensation of tryptamine with arginine. 3. The peptide families identified in skin extracts of the African frogs were as follows: caeruleins (caerulein, [Asn2, Leu5] caerulein), tachykinins (kassinin, [Glu2, Pro5] kassinin, hylambatin), bradykinins [( Hyp3] bradykinin), xenopsin, thyrotropin releasing hormone, peptide PYLa and the magainins I and II. The last five peptides have been so far identified only in the skin of Xenopus laevis, together with their precursors. 4. Since numerous other peptide molecules await isolation, elucidation of structure, and definition of possible biological activities, the array of peptides occurring in the skin of African amphibians, as in that of Australian and American amphibians, is destined to increase.     

Pharmacology of amphibian opiate peptides

In 1980 the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists, and named dermorphins. Since 1987 deltorphins, a family of highly selective delta-opioid peptides were identified either by cloning of the cDNA from frog skins or isolation of the peptides. The distinctive feature of opioid peptides is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe. The discovery of the amphibian opiate peptides, provided new insights into the functional role of the mu- and delta-opiate systems. It also provided models for novel analgesics with enhanced therapeutic benefits and reduced toxicity.     

Divergent landscape effects on population connectivity in two co-occurring amphibian species

The physical and environmental attributes of landscapes often shape patterns of population connectivity by influencing dispersal and gene flow. Landscape effects on movement are typically evaluated for single species. However, inferences from multiple species are required for multi‐species management strategies increasingly being applied in conservation. In this study, I compared the spatial genetic patterns of two amphibian species across the northeastern United States and estimated the influence of specific landscape features on the observed genetic structure. The spotted salamander (Ambystoma maculatum) and wood frog (Rana sylvatica) share many ecological attributes related to habitat use, phenology and site fidelity. However, I hypothesized that important differences in their movement patterns and life history would create distinct genetic patterns for each species. Using 14 microsatellite loci, I tested for differences in the level of genetic differentiation between the two species across 22 breeding ponds. The effects of eight landscape features were also estimated by evaluating 32 landscape resistance models. Spotted salamanders exhibited significantly higher genetic differentiation than wood frogs. Different landscape features were also identified as potential drivers of the genetic patterns in each species, with little overlap in model support between species. Collectively, these results provide strong evidence that these two amphibian species interact with the landscape in measurably different ways. The distinct genetic patterns observed are consistent with key differences in movement ability and life history between A. maculatum and R. sylvatica. These results highlight the importance of considering more than one species when assessing the impacts of the landscape matrix on population connectivity, even for ecologically similar species within the same habitats.     

Predator-prey interactions between two amphibian species: effects of insecticide exposure

The presence of environmental contaminants may alter predator-prey interactions among aquatic species by altering activity levels of predators or prey, or by altering predator avoidance behavior. The outcome of a predatory encounter may be dependent upon whether both species are exposed to a contaminant simultaneously, or whether exposure occurs only in one of the species. In a laboratory experiment, I used the insecticide carbaryl to examine predation of southern leopard frog tadpoles (Rana sphenocephala) by adult red-spotted newts (Notophthalmus viridescens) under four conditions: both tadpoles and newts exposed, neither tadpoles nor newts exposed, and either newts or tadpoles only exposed. After one hour, exposed newts consumed half as many tadpoles as non-exposed newts. Carbaryl potentially affected newt activity enough to reduce time spent searching for prey, or may have altered the speed and coordination necessary to capture tadpoles. After six hours, non-exposed and exposed newts consumed similar numbers of tadpoles, most likely indicating recovery from exposure. After 24 h, predation rates were lowest when both newts and tadpoles were simultaneously either exposed or not exposed, and were greatest when newts and tadpoles were not exposed simultaneously. This study suggests that when tadpoles and newts are exposed to a sublethal level of a contaminant simultaneously, that predation rates do not differ from those observed under natural conditions, but exposure of either predator or prey at different times can disrupt predator-prey dynamics. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic differentiation and species cohesion in two widespread Central American Begonia species

Begonia is one of the ten largest plant genera, with over 1500 species. This high species richness may in part be explained by weak species cohesion, which has allowed speciation by divergence in allopatry. In this study, we investigate species cohesion in the widespread Central American Begonia heracleifolia and Begonia nelumbiifolia, by genotyping populations at microsatellite loci. We then test for post-zygotic reproductive barriers using experimental crosses, and assess whether sterility barriers are related to intraspecific changes in genome size, indicating major genome restructuring between isolated populations. Strong population substructure was found for B. heracleifolia (F_ST=0.364, F′_ST=0.506) and B. nelumbiifolia (F_ST=0.277, F′_ST=0.439), and Bayesian admixture analysis supports the division of most populations into discrete genetic clusters. Moderate levels of inferred selfing (B. heracleifolia s=0.40, B. nelumbiifolia s=0.62) and dispersal limitation are likely to have contributed to significant genetic differentiation (B. heracleifolia Jost''s D=0.274; B. nelumbiifolia D=0.294). Interpopulation crosses involving a divergent B. heracleifolia population with a genome size ∼10% larger than the species mean had a ∼20% reduction in pollen viability compared with other outcrosses, supporting reproductive isolation being polymorphic within the species. The population genetic data suggest that Begonia populations are only weakly connected by gene flow, allowing reproductive barriers to accumulate between the most isolated populations. This supports allopatric divergence in situ being the precursor of speciation in Begonia, and may also be a common speciation mechanism in other tropical herbaceous plant groups.     

Presence of ranatensin-like and bombesin-like peptides in amphibian brains

Antibodies specific for the carboxyl-terminal regions of bombesin and of ranatensin were used to study the tissue distribution of substances immunochemically similar to these two peptides in three amphibian species. Brain extracts of Rana catesbeiana, Rana pipiens, and Xenopus laevis all contained considerable quantities of both types of immunoreactivity, with measured concentrations as high as several hundred pmol per g tissue. The two antibodies used in this study had very low crossreactivity (less than 1% by RIA) with the other peptide. In addition, gel filtration revealed different elution profiles for the two immunoreactive substances extracted from amphibian brains. Immunocytochemistry revealed differences in localization within nerve fibers and cell bodies and specific absorption by the appropriate peptide. Ranatensin-like peptides were also present in high concentrations in skin of the two Rana species but not in that of Xenopus. Bombesin-like peptides were more abundant in the stomach of all three species. Significant amounts of substance P/phylasaemin-like immunoreactivity also were detected in the brains of all three species. It is concluded that ranatensin-like peptides are not confined to the skin and can be included as central nervous system neuropeptides in amphibians. These two groups of peptides are not species-specific since both are found in brain and stomach of amphibians whose skin contains only ranatensin-like peptide or neither.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

Effects of supraspinal administration of PG-SPI and PG-KII, two amphibian tachykinin peptides, on nociception in the rat

We investigated and compared the effects of two amphibian tachykinins, the NK1 receptor agonist PG-SPI and the NK3 receptor agonist PG-KII, and the mammalian tachykinins substance P, neurokinin A and neurokinin B on the reaction time to a painful radiant heat stimulus (tail-flick test in rats) after intracerebroventricular injection. PG-SPI (1, 10 and 20 microg) and PG-KII (1, 5 and 10 microg) significantly increased the reaction time. Substance P (10 microg) injected intracerebroventricularly induced antinociception, whereas neurokinin A and neurokinin B did not. Like analgesia evoked by exogenous substance P, PG-SPI-evoked analgesia was blocked by pretreatment with naloxone. Naloxone left PG-KII antinociception unchanged, but the NK3 receptor selective antagonist markedly reduced it. These findings suggest NK1 and NK3 tachykinin receptor system involvement in supraspinal analgesia in rats.     

Analysis of fatty acid composition revealed differences in the diets of tadpoles of two amphibian species

The study of the composition of fatty acid markers of tadpoles of cohabiting amphibian species for the first time revealed differences in their diets: the moor frog Rana arvalis prefers bacteria not associated with plant detritus, whereas the diet of the common spadefoot Pelobates fuscus is based on cyanobacteria, green algae, diatoms, and possibly higher plants. Major differences in the fatty acid composition are determined by the difference in the percentage of eicosapentaenoic and myristic acids.     

Active proton and urea transport by amphibian skin

Proton secretion in frog skin is mediated by an electrogenic H+ pump. Pharmacological and immunocytological approaches have identified this pump as belonging to the V-ATPase class. The key role of this V-ATPase in proton secretion (acid-base balance) and as a membrane energizer of other solute transport from very dilute solutions is outlined. It is shown that the frog skin constitutes a model of a V-ATPase-dependent Na+ transport mechanism applicable to other freshwater animals. On the other hand, attempts to implicate the V-ATPase in the active urea transport that develops through the skin of salt-adapted frogs have failed; the nature of the different urea transporters located on apical and basal epithelial cell membranes and those responsible for active urea reabsorption remain to be identified.     

Cranial suture closure in two species of South American monkeys

Cranial suture closure is examined in two species of South American monkeys, Saimiri sciureus and Saguinus nigricollis. Sequences in closure were sought as indicators of skeletal age. Some sutures seem to be more reliable determinants of skeletal age than others, and these sutures and their sequence of closure are different in the two species examined. The sphenooccipital synchondrosis and the palatal portion of the Interpremaxillary suture show regular fusion associated with age in both species. In Saimiri the maxillopremaxillary sutures are also reliable indicators of age, whereas they are not in Saguinus; however, in the latter the presphenoid-postsphenoid synchondrosis closes regularly whereas it does not in Saimiri. In Saimiri the predictable sequence is (1) maxillo-premaxillary, (2) transverse maxillo-premaxillary, (3) spheno-occipital, (4) interpremaxillary. In Saguinus it is (1) presphenoid-postsphenoid, (2) spheno-occipital, (3) interpremaxillary. It is possible that the sequences of suture closure and the variability in this process may indicate genetic and taxonomic relationships.