Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs Litoria caerulea with severe chytridiomycosis

Although histopathology is used routinely for diagnosis of chytridiomycosis in live and dead amphibians, there are no quantitative data on the distribution of the causative fungus, Batrachochytrium dendrobatidis, in the skin. We performed quantitative histological examinations on 6 sites on the body and 4 toes of 10 free-ranging adult green tree frogs Litoria caerulea found recently dead or dying from chytridiomycosis. Large numbers of sporangia occurred in all areas of ventral skin and toes; on average there were 94.3 sporangia mm(-1) of superficial epidermis. The number of sporangia was highly variable and this appeared to be related to the stage in the cycle of sloughing. The stratum corneum tends to build up with high intensities of infection and then sheds entirely rather than being shed continuously. Very few or no sporangia occurred on dorsal skin. This distribution could be explained by the dryness of the dorsal skin or possibly by the greater number of serous glands, which produce antifungal peptides, on the dorsum. In some frogs, ulceration and erosions occurred on skin on the back in the absence of sporangia. Other pathological changes such as hyperkeratosis and congestion occurred much more frequently on ventral surfaces.     

Surface movement in water of splendipherin, the aquatic male sex pheromone of the tree frog Litoria splendida

The aquatic sex pheromone splendipherin (GLVSSIGKALGGLLADVVKSKGQPA-OH) of the male green tree frog Litoria splendida moves across the surface of water to reach the female. Surface pressure and X-ray reflectometry measurements confirm that splendipherin is a surface-active molecule, and are consistent with it having an ordered structure, whereby the hydrophilic portion of the peptide interacts with the underlying water and the hydrophobic region is adjacent to the vapour phase. The movement of splendipherin over the surface of water is caused by a surface pressure gradient. In order to better define the structure of splendipherin at the water/air interface we used 2D NMR studies of the pheromone with the solvent system trifluoroethanol/water (1 : 1 v/v). In this solvent system, splendipherin adopts a bent alpha helix from residues V3 to K21. The bending of the helix occurs in the centre of the peptide in the vicinity of G11 and G12. The region of splendipherin from V3 to G11 has well-defined amphipathicity, whereas the amphipathicity from G12 to A25 is reduced by K19 and P24 intruding into the hydrophobic and hydrophilic regions respectively. A helical structure is consistent with X-ray reflectometry data.     

Differences in the skin peptides of the male and female Australian tree frog Litoria splendida. The discovery of the aquatic male sex pheromone splendipherin, together with phe8 caerulein and a new antibiotic peptide caerin 1.10.

The skin secretions of female and male Litoria splendida have been monitored monthly over a three-year period using HPLC and electrospray mass spectrometry. Two minor peptides are present only in the skin secretion of the male. The first of these is the female-attracting aquatic male sex pheromone that we have named splendipherin, a 25 amino acid peptide (GLVSSIGKALGGLLADVVKSKGQPA-OH). This pheromone constitutes about 1% of the total skin peptides during the breeding season (January to March), dropping to about 0.1% during the period June to November. Splendipherin attracts the female in water at a concentration of 10-11-10-9 M, and is species specific. The second peptide is a wide-spectrum antibiotic of the caerin 1 group, a 25 residue peptide (GLLSVLGSVAKHVLPHVVPVIAEKL-NH2) named caerin 1.10. The neuropeptides of L. splendida are also seasonally variable, the change identical for both the female and male. During the period October to March, the sole neuropeptide present in skin secretions is caerulein [pEQDY(SO3)TGWMDF-NH2]; this is active on smooth muscle and is also an analgaesic. During the southern winter (June to September), more than half of the caerulein is hydrolysed to [pEQDYTGWMDF-NH2], a peptide that shows no smooth muscle activity. In place of caerulein, a new peptide, Phe8 caerulein [pEQDY(SO3)TGWFDF-NH2], becomes a major component of the skin secretion. Perhaps this seasonal change is involved in thermoregulation, that is, with the initiation and maintenance of the inactive (hibernation) phase of the animal.     

Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs

During the breeding season of the mountain chicken frog Leptodactylus fallax, fighting between males results in the emergence of dominant animals that subsequently attract females to nesting sites. A peptide, termed Leptodactylus aggression-stimulating peptide (LASP), was isolated from norepinephrine-stimulated skin secretions from male specimens of L. fallax that was not present in skin secretions obtained from females. The primary structure of the peptide was established as: Gly-Leu-Trp-Asp-Asp-Leu-Lys-Ala-Ala-Ala-Lys-Lys-Val-Val-Ser-Ser-Leu-Ala-Ser-Ala-Ala-Ile-Glu-Lys-Leu NH2. LASP had no pheromone-like action on females but had a chemoattractive effect on males and stimulated aggressive behaviors, such as rearing and leaping. It is suggested that this peptide may play an important role in initiating the competitive male-male interactions that are associated with the onset of reproductive behavior in L. fallax.     

Isolation, structural characterization, and bioactivity of a novel neuromedin U analog from the defensive skin secretion of the Australasian tree frog, Litoria caerulea

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M(r) 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Light and electron microscopic analyses of the high deformability of adhesive toe pads in White's tree frog,Litoria caerulea

White's tree frog (Litoria caerulea) has large, adhesive toe pads that are among the softest of all known biological structures. To explore the morphological basis for the physical properties of the toe pads, the internal microstructure of the toe pads in L. caerulea was examined using both light and transmission electron microscopy. Three design elements that are distinct from other areas of skin were observed. First, the keratinocytes comprising the adhesive surface of the toe pad all contained keratin filament bundles (tonofibrils) exhibiting structural anisotropy. Specifically, the curved conformation of the hierarchical (branching) tonofibrils was characterized by the formation of anastomoses consisting of tonofibrils beneath the adhesive cell surface and stem keratin filament bundles concentrated in the lower‐middle part of the dorsal‐side of adhesive cells. Second, the cytoplasm of keratinocytes in the most superficial cell layer contained glycoproteins (stained by periodic acid/Schiff reagent) that are considered to confer high viscoelasticity. Third, the dermis contained large lymph spaces interspersed with elastic fibers and collagen fibers, which were relatively sparsely distributed compared to the dorsal skin of the toe pads. The profiles of these structures were easily deformed by the slight application of pressure. These findings reaffirmed that the unique internal architecture of the toe pads in L. caerulea contributed to their remarkable softness and high deformability, which in turn increased the contact area and provided improved adaptability to the local topography of natural surfaces. J. Morphol. 277:1509–1516, 2016. © 2016 Wiley Periodicals, Inc.     

Comparison of isometric contractile properties of the tongue muscles in three species of frogs, Litoria caerulea, Dyscophus guinetti, and Bufo marinus.

Previous studies show that anurans feed in at least three different ways. Basal frogs have a broad tongue that shortens during protraction and emerges only a short distance from the mouth. Some frogs have long, narrow tongues that elongate dramatically due primarily to inertia from mouth opening, which is transferred to the tongue. A few species have a hydrostatic mechanism that produces tongue elongation during protraction. This functional diversity occurs among frogs that share the same two pairs of tongue muscles. Our study compares the isometric contractile properties of these tongue muscles among three frog species that represent each feeding mechanism. Nerves to the paired protractors and retractors were stimulated electrically in each species to record the force properties, contraction speeds, and fatigabilites of these muscles. Few differences were found in the isometric contractile properties of tongue muscles, and the greatest differences were found in the retractors, not the protractors. We propose that the unique arrangement of the tongue muscles in frogs results in a retractor that may also be coactivated with the protractor in order to produce normal tongue protraction. Inertial effects from body, head, and jaw movements, along with clear differences that we found in passive resistance of the tongues to elongation, may explain much of the behavioral variation in tongue use among species.     

Spatial distributions of male and female strawberry poison frogs and their relation to female reproductive resources

In many species with a resource-based mating system, males defend resources to increase their attractiveness to females. In the strawberry poison frog, Dendrobates pumilio, suitable tadpole-rearing sites appear to be a limited resource for females. Territorial males have been suggested to defend tadpole-rearing sites to increase their access to females. In this study we investigate the spatial association between tadpole-rearing sites and the sexes as well as the spatial association of males and females. If strawberry poison frogs have resource defense polygyny, we expect males and females to be associated with tadpole-rearing sites and that females will deposit their offspring in tadpole-rearing sites inside the territories of their mates. To test this hypothesis, home range and core area sizes were calculated for both sexes and the association patterns were compared in two areas that differed in their abundance of tadpole-rearing sites. Home ranges and core areas of females were much larger than male home ranges. Females showed a clumped distribution in the vicinity of tadpole-rearing sites. Males were not clumped and were less associated with tadpole-rearing sites. Females generally did not use tadpole-rearing sites in the territory of their mates and we therefore conclude that males did not defend tadpole-rearing sites for females. Our data are consistent with the general assumption that female distribution is influenced by resource distribution and that male distribution depends on female distribution. Nevertheless, the distribution of D. pumilio females was also influenced by male spacing patterns. Males probably initially establish their core areas where female density is high and then females move among territories to sample males. Males compete vigorously for places with high female density, the defense of which is likely important for enhancing their mating success. In general, the spacing patterns did not differ between populations but the sex ratio was strongly female biased in the habitat with more tadpole-rearing sites, reflecting the direct reliance of females on these resources.     

Molecular patterns of introgression in a classic hybrid zone between the Australian tree frogs,Litoria ewingii and L. paraewingi: evidence of a tension zone

Hybrid zones provide a rare opportunity to explore the processes involved in reproductive isolation and speciation. The southern hybrid zone between the southeastern Australian tree frogs Litoria ewingii and L. paraewingi has been comprehensively studied over the last 40 years, primarily using reproductive compatibility experiments and male advertisement calls. We used mitochondrial DNA (mtDNA) and eight nuclear microsatellite markers to characterize this hybrid zone along a historically studied transect and to test various dispersal‐dependent and dispersal‐independent hybrid zone models. The species are genetically distinct and the level of hybridization within the contact zone is low, with the majority of admixed individuals representing later‐generation hybrids. Based on previous experimental genetic compatibility studies, we predicted that hybrids with L. paraewingi mtDNA would be more frequent than hybrids with L. ewingii mtDNA. Surprisingly, a greater proportion of the identified hybrids had L. ewingii mtDNA. Geographical cline analyses showed a sharp transition in allele frequencies across the transect, and both the mtDNA and microsatellite data showed concordant cline centres, but were best supported by a model that allowed width to vary. Overall, the L. ewingii–L. paraewingi hybrid zone is best characterized as a tension zone, due to the narrow cline width, concordant genetic clines and low levels of hybridization.     

Male and female effects on fertilization success and offspring viability in the Peron's tree frog,Litoria peronii

Abstract There is increasing theoretical and empirical evidence that genetic compatibility among partners is an important determinant of fertilization success and offspring viability. In amphibians, females often actively choose partners from among a variety of males and polyandry is common. Genetic compatibility among partners may therefore be an important determinant of fertilization success and offspring viability in some amphibians. Amphibians also show some of the highest levels of genetic differentiation among neighbouring populations known in vertebrates, and as such, populations may have evolved different co‐adapted gene complexes. This means that offspring from among‐population crosses may have reduced fitness. It is therefore essential to understand to what extent crossings between and within populations may interfere with successful fertilization and offspring viability. Here, we test whether crossing individuals within and between two different populations of the Australian Peron's tree frog (Litoria peronii) using artificial fertilizations affect fertilization success and offspring viability. Fertilization success per se is strongly influenced by male identity, which is likely to depend at least to some extent on the experimental procedure (e.g. resulting in variation in sperm number per ejaculate), whereas there was no fertilization effect of female identity. More importantly, male and female identity, independently of each other, explained significant variation in offspring viability, whereas no such effect could be linked to population of origin. Thus, our experiments suggest that crossing populations may not always be the most significant factor affecting fertilization success or offspring viability, but may be more influenced by the genetic quality or the genetic compatibility of partners.     

Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata solution structure and biological activity.

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.     

Peptidomic analysis of skin secretions supports separate species status for the tailed frogs,Ascaphus truei and Ascaphus montanus

The tailed frog Ascaphus truei Stejneger, 1899 is the most primitive extant anuran and the sister taxon to the clade of all other living frogs. The species occupies two disjunct ranges in the Northwest region of North America: the Cascade Mountains and coastal area from British Columbia to Northern California, and an inland range in the northern Rocky Mountains and the Blue and Wallowa mountains. A previous study led to the isolation of eight peptides with antimicrobial activity (termed the ascaphins) from skin secretions of A. truei from the coastal range. The present study has used peptidomic analysis to identify the products of orthologous ascaphin genes in electrically-stimulated skin secretions from inland range specimens. Structural characterization of the peptides demonstrated that ascaphins from the inland range contained the following amino acid substitutions compared with orthologs from the coastal range frogs: ascaphin-1 (Ala¹² → Glu), ascaphin-3 (Asp⁴ → Glu), ascaphin-4 (Ala¹⁹ → Ser), ascaphin-5 (Lys¹² → Thr), and ascaphin-7 (Gly⁸ → Ser and Ser²⁰ → Asn). Orthologs of ascaphins-2, -6, and -8 were not identified but a paralog of ascaphin-5, identical to ascaphin-5 from coastal range frogs, was found. The data support the claims, derived from analysis of the nucleotide sequences of mitochondrial genes, that the inland populations of the tailed frog should be recognized as a distinct species, the Rocky Mountain tailed frog Ascaphus montanus and that the divergence of the species from A. truei probably occurred in the late Miocene (approximately 10 Mya).     

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata.

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus.     

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly¹³ → Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.