Gastric brooding: unique form of parental care in an Australian frog.

The recently described leptodactylid frog Rheobatrachus silus of Queensland, Australia, exhibits a unique form of parental care. The female carries embryos and young in the stomach, propulsively ejecting the juveniles.

Inhibition of gastric acid secretion in the gastric brooding frog, Rheobatrachus silus.

The female gastric brooding frog Rheobatrachus silus broods its young in its stomach. A substance that inhibits gastric acid secretion in a toad stomach preparation in vitro appears to be secreted by the developing young. This substance has been identified as prostaglandin E2. Rheobatrachus silus may thus have developed a mechanism whereby prostaglandin secreted by the larvae inhibits acid secretion in the stomach of the female until the larvae have completed development and emerged as juvenile frogs by way of the female's mouth.

Superficial mandibular musculature and vocal sac structure in hemiphractine hylid frogs.

Three patterns of superficial mandibular musculature are described in hemiphractine hylid frogs. One of these is unique to the morphologically bizarre Hemiphractus. A second pattern is found in Flectonotus and also occurs in some species of Gastrotheca and Stefania. A third pattern involves a differentiated apical element of the m. intermandibularis and is found in Cryptobatrachus, many species of Gastrotheca, and one species of Stefania. Evidence supports the plesiomorphic state of an undifferentiated m. intermandibularis and two derived states of differentiation of that muscle. One of these is the development of supplementary posterolateral elements characteristic of the Phyllomedusinae, whereas the differentiation of an apical element has occurred in at least six independent lineages--the entire Pelodryadinae, three unrelated genera of Hylinae, and two genera of Hemiphractinae. Gastrotheca and Stefania are the only anuran genera known to include species possessing, and others lacking, differentiation of the m. intermandibularis. Vocal sacs and apertures are absent in Cryptobatrachus, Hemiphractus, Stefania, and six species of Gastrotheca.

Effect of season and temperature on mortality in amphibians due to chytridiomycosis.

OBJECTIVE: To investigate the distribution and incidence of chytridiomycosis in eastern Australian frogs and to examine the effects of temperature on this disease. DESIGN: A pathological survey and a transmission experiment were conducted. PROCEDURE: Diagnostic pathology examinations were performed on free-living and captive, ill and dead amphibians collected opportunistically from eastern Australia between October 1993 and December 2000. We conducted a transmission experiment in the laboratory to investigate the effects of temperature: eight great barred frogs (Mixophyes fasciolatus) exposed to zoospores of Batrachochytrium dendrobatidis and six unexposed frogs were housed individually in each of three rooms held at 17 degrees C, 23 degrees C and 27 degrees C. RESULTS: Chytridiomycosis was the cause of death or morbidity for 133 (55.2%) of 241 free-living amphibians and for 66 (58.4%) of 113 captive amphibians. This disease occurred in 34 amphibian species, was widespread around the eastern seaboard of Australia and affected amphibians in a variety of habitats at high and low altitudes on or between the Great Dividing Range and the coast. The incidence of chytridiomycosis was higher in winter, with 53% of wild frogs from Queensland and New South Wales dying in July and August. Other diseases were much less common and were detected mostly in spring and summer. In experimental infections, lower temperatures enhanced the pathogenicity of B. dendrobatidis in M. fasciolatus. All 16 frogs exposed to B. dendrobatidis at 17 degrees C and 23 degrees C died, whereas 4 of 8 frogs exposed at 27 degrees C survived. However, the time until death for the frogs that died at 27 degrees C was shorter than at the lower temperatures. Infections in survivors were eliminated by 98 days. CONCLUSION: Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.

The submandibular musculature of Phyllomedusinae (Anura: Hylidae): A reappraisal.

The submandibular musculature of 37 species of the five currently recognized genera of the subfamily Phyllomedusinae (Anura: Hylidae) is described; observations are made on the variation and ontogeny of these muscles. Supplementary apical elements of the m. intermandibularis occur in all phyllomedusines studied, in addition to the supplementary posterolateral elements previously reported. Our observations are discussed in the context of 1) the proposed homology between supplementary apical and posterolateral elements; 2) the homology with the apical elements reported for Pelodryadinae (sister taxon of Phyllomedusinae); and 3) the implications for our understanding of the relationships between Phyllomedusinae and Pelodryadinae. Anatomical differences between the apical and posterolateral elements and their co-occurrence in phyllomedusines indicate that these supplementary elements are not homologous. Despite differences between phyllomedusines and pelodryadines in the adhesion of supplementary fibers to the principal element of the m. intermandibularis and the occurrence of a broad aponeurosis or a medial raphe, the extensive morphological and developmental resemblances of the apical elements indicate that these structures are homologous, and that the presence of apical elements is a synapomorphy of Phyllomedusinae + Pelodryadinae.

Mosquito repellents in frog skin.

The search for novel insect repellents has been driven by health concerns over established synthetic compounds such as diethyl-m-toluamide (DEET). Given the diversity of compounds known from frog skin and records of mosquito bite and ectoparasite infestation, the presence of mosquito repellents in frogs seemed plausible. We investigated frog skin secretions to confirm the existence of mosquito repellent properties. Litoria caerulea secretions were assessed for mosquito repellency by topical application on mice. The secretions provided protection against host-seeking Culex annulirostris mosquitoes. Olfactometer tests using aqueous washes of skin secretions from L. caerulea and four other frog species were conducted to determine whether volatile components were responsible for repellency. Volatiles from Litoria rubella and Uperoleia mjobergi secretions were repellent to C. annulirostris, albeit not as repellent as a DEET control. The demonstration of endogenous insect repellents in amphibians is novel, and demonstrates that many aspects of frog chemical ecology remain unexplored.

Pepsin in the toad Bufo marinus.

The macroscopic localization of pepsinogen in the upper gastrointestinal tract of the anuran Bufo marinus was studied by means of biochemical assay. The pH optimum of the anuran pepsin was determined to be 1.6. The effect of lowered ambient temperature on stored pepsinogen and proteolytic activity was studied. Both parameters were reduced. The results are extrapolated to speculate on the mechanisms of gastric brooding in gastric brooding frogs of the genus Rheobatrachus.

Changes in the organization and ultrastructure of smooth muscle cells in the stomach of the gastric brooding frog, Rheobatrachus silus, during brooding.

The young of the aquatic Australian frog, Rheobatrachus silus (Leptodactylidae) develop from eggs to juvenile frogs in the mother's stomach. During brooding the stomach expands greatly and becomes very thin walled. Transmission electron microscopy showed that the distension of the stomach was accompanied by a severe disruption of the smooth muscle layers. Many of the smooth muscle cells seemed to be highly contracted and resembled smooth muscle cells contracted in the absence of an intact connective tissue matrix. Eight days after the birth of the juveniles through the mouth of the female, the stomach muscle cells had returned to a normal appearance. It is suggested that during gastric incubation of the young, smooth muscle cells become at least partially dissociated from their surrounding connective tissue matrix, allowing maximal distension of the stomach wall.

Gastric acid secretion in the toad Bufo marinus with the description of a new technique for in vivo monitoring.

A technique for the implantation of a pH electrode into the stomach of the toad, Bufo marinus, is described. The technique permits continuous monitoring of gastric pH for extended periods. Results from continuous monitoring and from acute measurements of gastric pH are compared in fasting and fed toads. Prostaglandin E, given orally with food, caused a dose-related delay in the fall of gastric pH which followed feeding.

Converting a stomach to a uterus: the microscopic structure of the stomach of the gastric brooding frog Rheobatrachus silus.

Rheobatrachus silus is a rare aquatic frog of eastern Australia. The female ingests the eggs after fertilization and broods them in the stomach until fully formed. "Gastric brooding" takes place in the fundus and proximal part of the body of the stomach, which dilates to accommodate the growing young. The surface epithelium becomes attenuated and the cells contain fewer mucus droplets. The acini of the glands are less numerous because of stretching, and they contain oxyntic cells that show evidence of profound suppression or regression. Morphometric studies on the limited number of samples show that the oxyntic cells are attenuated with few surface projections, sparse tubulovesicular reticulum, and a few pepsinogen granules and mitochondria. Eight days after ejection of the young, and 4 days after feeding commences, the lining shows a return of gastric pits and glands. The oxyntic cells show many surface projections and a proliferation of the tubulovesicular reticulum and mitochondria. These findings suggest that the eggs, tadpoles, and juvenile frogs release a substance, or substances, that inhibit acid secretion immediately after the eggs are ingested and that persist throughout brooding.

Caerulein-like peptides from the skin glands of the Australian Blue Mountains tree frog Litoria citropa. Part 1. Sequence determination using electrospray mass spectrometry.

Sixteen caerulein-type peptides have been isolated from the skin secretions of the Australian Blue Mountains tree frog Litoria citropa. There are four groups of these peptides. The first is based on the structure of the known neuropeptide caerulein [pEQDY(SO(3))TGWMDF-NH(2)], now renamed caerulein 1.1. Examples of peptides of the other groups are as follows: caerulein 2.1 [pEQDY(SO(3))TGAHMDF-NH(2)], caerulein 3.1 [pEQDY(SO(3))GTGWMDF-NH(2)] and caerulein 4.1 [pEQDY(SO(3))TGSHMDF-NH(2)]. All of these peptides are accompanied by the associated peptide where Phe replaces Met, and all eight of the caerulein peptides are accompanied by the desulfated analogues. Negative ion electrospray mass spectrometry (ES-MS) is used to determine the molecular weights of the caeruleins 1-4 [from their [M - H](-) ions], while the sequences of the peptides are determined from the B and Y + 2 cleavage ions in the mass spectra of the [MH(+) - SO(3)](+) ions.

Gastric emptying and intestinal transit in Bufo marinus and the actions of E prostaglandins.

Gastric emptying and intestinal transit have been studied in the anuran Bufo marinus using Tc99-labelled chicken liver as the test meal. Gastric emptying was measured as the proportion of the test meal remaining in the stomach after 3 or 6 h. Intestinal transit was calculated by the geometric centre method at the same time intervals. There was a high degree of variability in these parameters between individual animals, in both control and treated groups. Both SC29333 (Searle), a synthetic E prostaglandin, and prostaglandin E2 given orally delayed gastric emptying. These results complement the finding that prostaglandin E2 causes relaxation of anuran longitudinal muscle in vitro, and lend further support to the suggestion that prostaglandin E2 may be responsible for the gut stasis associated with gastric brooding in the gastric brooding frog Rheobatrachus silus.

The application of mass spectrometry to the study of evolutionary trends in amphibians.

The glandular secretions of the skin of Litoria rubella specimens collected from five locations on the eastern seaboard of Queensland (Australia) contain the three tryptophyllin peptides Phe Pro Trp Leu (NH2), Phe Pro Trp Pro (NH2) and Phe Pro Phe Pro Trp Leu (NH2). The relative proportions of these peptides in the glandular secretion are associated with geographic location, i.e. Phe Pro Trp Pro (NH2) is a minor component of the peptide mixture in frogs from southern Queensland, but becomes significantly more abundant as the location becomes more northerly. This trend indicates an evolutionary change in the animal, but for what reason, and over what timescale is not known at this time.

New caerin antibacterial peptides from the skin glands of the Australian tree frog Litoria xanthomera. Part 2. Sequence determination using mass spectrometry and associated techniques.

Mass spectrometric sequencing, enzymic digestion and Edman degradation provide the structures of the two antimicrobial peptides from the skin glands of the Australian tree frog Litoria xanthomera as:- Gly Leu Phe Ser Val Leu Gly Ala Val Ala Lys His Val Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2) (caerin 1.6), and Gly Leu Phe Lys Val Leu Gly Ser Val Ala Lys His Leu Leu Pro His Val Ala Pro Val Ile Ala Glu Lys Leu (NH2) (caerin 1.7).

The citropin peptides from the skin glands of the Australian Blue Mountains tree frog Litoria citropa. Part 2: sequence determination using electrospray mass spectrometry.

A combination of electrospray mass spectrometry, Lys-C digest/mass spectrometry and automated Edman sequencing provides the amino acid sequences of nineteen citropin peptides isolated from the granular dorsal and submental glands of the Blue Mountains tree frog Litoria citropa. Citropin 1.1 [Gly Leu Phe Asp Val Ile Lys Lys Val Ala Ser Val Ile Gly Gly Leu (NH(2))] and citropin 1.2 [Gly Leu Phe Asp Ile Ile Lys Lys Val Ala Ser Val Val Gly Gly Leu (NH(2))] are the two major skin peptides: both show significant wide-spectrum antibacterial activity.

First record of host defence peptides in tadpoles. The magnificent tree frog Litoria splendida.

Tadpoles of the Magnificent Tree Frog Litoria splendida produce host defence peptides early in their development and well before metamorphosis. Peptides were identified and characterized using high performance liquid chromatography and electrospray mass spectrometry. No host defence peptides were identified in the eggs. The neuropeptide caerulein was detected 10 d after egg deposition, and the antibiotic peptides caerin 1.1, caerin 1.6 and caerin 3.1 first appeared at 14 d. The concentration of peptides increases with the onset of metamorphosis at 84 d, when the host-defence peptide profile is the same as that of the adult.

Linguistically mediated visual search.

During an individual's normal interaction with the environment and other humans, visual and linguistic signals often coincide and can be integrated very quickly. This has been clearly demonstrated in recent eye tracking studies showing that visual perception constrains on-line comprehension of spoken language. In a modified visual search task, we found the inverse, that real-time language comprehension can also constrain visual perception. In standard visual search tasks, the number of distractors in the display strongly affects search time for a target defined by a conjunction of features, but not for a target defined by a single feature. However we found that when a conjunction target was identified by a spoken instruction presented concurrently with the visual display, the incremental processing of spoken language allowed the search process to proceed in a manner considerably less affected by the number of distractors. These results suggest that perceptual systems specializedfor language and for vision interact more fluidly than previously thought.

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis. Part 2. Sequence determination using electrospray mass spectrometry.

Sixteen aurein peptides are present in the host defence secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aureus and seventeen from those of the related Southern Bell Frog Litoria raniformis. All peptides have been sequenced using a combination of electrospray mass spectrometry and Lys-C digestion, with each sequence confirmed by automated Edman sequencing. The peptides are named in five groups, viz. aureins 1-5. Ten of these peptides are common to both species of frog. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), the smallest peptide from an anuran reported to have both antibiotic and anticancer activity; aurein 2.2 (GLLDIVKKVIGAFGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). The aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH), show neither antibacterial nor anticancer activity.

New caerin antibacterial peptides from the skin glands of the Australian tree frog Litoria xanthomera.

The secretion of the skin glands of the 'orange-thighed frog' Litoria xanthomera contains seven peptides. One of these is the know hypotensive peptide caerulein. Two new peptides, caerin 1.6 [GLFSVLGAVAKHVLPHVVPVIAEKL(NH2)], and caerin 1.7 [GLFKVLGSVAKHLLPHVAPVIAEKL(NH2)] show antibacterial properties. Two other peptides lack the first two amino acid residues of caerins 1.6 and 1.7 and show no antibacterial activity. The identification of the peptides in Litoria xanthomera confirms that this species is related to Litoria caerula, Litoria gilleni and Litoria splendida but not as closely as those three species are related to each other.