Hatching timing,oxygen availability,and external gill regression in the tree frog,Agalychnis callidryas

The physiological role of the embryonic external gills in anurans is equivocal. In some species, diffusion alone is clearly sufficient to supply oxygen throughout the embryonic period. In others, morphological elaboration and environmental regulation of the external gills suggest functional importance. Since oxygen stress is a common trigger of hatching, I examined the relationships among hatching timing, oxygen stress, and external gill loss. I worked with the red-eyed tree frog, Agalychnis callidryas, a species with arboreal eggs and aquatic tadpoles in which gill regression is associated with hatching, and hatching timing affects posthatching survival with aquatic predators. Both exposure to a hypoxic gas mixture and submergence in water, a natural context in which hypoxic stress can occur, induced early hatching. Exposure to hyperoxic gas mixtures induced regression of external gills, and subsequent exposure to air induced early hatching. Prostaglandin-induced external gill regression also induced hatching, and this effect was partially ameliorated by exposure to hyperoxic gas. Together, these results suggest that external gills enhance the oxygen uptake of embryos and are necessary to extend embryonic development past the onset of hatching competence.     

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.     

Development, surface exposure, and embryo behavior affect oxygen levels in eggs of the red-eyed treefrog, Agalychnis callidryas

Oxygen stress can slow development, induce hatching, and kill eggs. Terrestrial anamniote embryos face a potential conflict between oxygen uptake and water loss. We measured oxygen levels within eggs to characterize the respiratory environment for embryos of the red-eyed treefrog, Agalychnis callidryas, a Neotropical frog with arboreal egg masses and plastic hatching timing. Perivitelline oxygen partial pressure (Po2) was extremely variable both within and among eggs. Po2 increased with air-exposed surface of the egg and declined over the developmental period before hatching competence. Through the plastic hatching period, however, average Po2 was stable despite continued rapid development. Development was synchronous across a wide range of perivitelline Po2 (0.5-16.5 kPa), and hatching-competent embryos tolerated Po2 as low as 0.5 kPa without hatching. The variation in Po2 measured over short periods of time within individual eggs was as great as that measured across development or surface exposure, including sharp transients associated with embryo movements. There was also a strong gradient of Po2 across the egg from superficial to deep positions. Ciliary circulation of fluid within the egg is clearly insufficient to keep it mixed. Embryos may maintain development under hypoxic conditions by strategic positioning of respiratory surfaces, particularly external gills, to exploit the patchy distribution of oxygen within their eggs.     

Mating Patterns of Red-Eyed Treefrogs, Agalychnis callidryas and A. moreletii

Deviations from random mating in frogs are often explained by two different size‐based patterns. The large‐male mating advantage predicts that males found in amplexus with females will be larger on average than non‐amplectant males, whereas size‐assortative mating predicts that males and females found in amplexus will maintain an optimal size ratio. Both these pairing patterns are consistent with a female mating preference for larger males, or for males of a given size relative to the choosy female. I examined pairing patterns of two species of Neotropical hylids, Agalychnis callidryas and A. moreletii for three consecutive breeding seasons in Belize, Central America to evaluate whether mating behavior was influenced by either a large‐male mating advantage or size‐assortative mating. For each species, I compared size traits between amplectant and non‐amplectant males, and within amplectant pairs, and I quantified fertilization success for each amplectant pair. For both species I found evidence of deviations from random mating by size, but the nature of the deviations varied between species and among years. The proportion of eggs fertilized was consistently high among years for both species and there was no relationship between fertilization success and the size ratio of amplectant pairs. These data are consistent with female mate preference, but a role for male–male competition cannot be excluded. My findings suggest that mating patterns may be density‐dependent and that the nature and intensity of sexual selection may be increased by extreme environmental conditions.     

The development of behavioral defenses: a mechanistic analysis of vulnerability in red-eyed tree frog hatchlings

I examined the development and effectiveness of behavioral defensesof red-eyed tree frog hatchlings, Agalychnis callidryas, against predatoryshrimp Macrobrachium americanum. Arboreal eggs of A. callidryashatch early if attacked by egg predators and later if undisturbed,producing tadpoles that enter the water at different developmentalstages. Older hatchlings survive better than young hatchlings withaquatic predators, including shrimp. Hatchlings respond to shrimpby both increasing activity and avoiding the bottom microhabitat.Older hatchlings are more active and, in the presence of shrimp,avoid the bottom more than young hatchlings. These ontogeneticchanges in behavior improve survival. Specifically, the likelihoodof fleeing from shrimp increases with hatchling age, and fleeingis an effective defense. In contrast to results from experimentswith odonates, immobility does not reduce risk of shrimp attack, thusthere is no trade-off between fleeing and motionless crypsis.Shrimp spend most of their time on the bottom, where attacksare more often successful. Avoidance of the bottom microhabitatby tadpoles therefore improves survival. Evasive maneuvers alsofunction in defense, but evasiveness does not change with age.Morphology may limit microhabitat use by younger hatchlings.Failure to flee may reflect unresponsiveness to disturbance,which would reduce unnecessarily early hatching and limit exposureof young A. callidryas to aquatic predators.     

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog,Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.     

The effects of acute and developmental temperature on burst swimming speed and myofibrillar ATPase activity in tadpoles of the Pacific tree frog, Hyla regilla

The effects of acute and developmental temperature on maximum burst swimming speed, body size, and myofibrillar ATPase activity were assessed in tadpoles of the Pacific tree frog, Hyla regilla. Tadpoles from field-collected egg masses were reared in the laboratory at 15 degrees (cool) and 25 degrees C (warm). Body size, maximum burst swimming speed from 5 degrees to 35 degrees C, and tail myofibrillar ATPase activity at 15 degrees and 25 degrees C were measured at a single developmental stage. Burst speed of both groups of tadpoles was strongly affected by test temperature (P<0. 001). Performance maxima spanned test temperatures of 15 degrees -25 degrees C for the cool group and 15 degrees -30 degrees C for the warm group. Burst speed also depended on developmental temperature (P<0.001), even after accounting for variation in body size. At most test temperatures, the cool-reared tadpoles swam faster than the warm-reared tadpoles. Myofibrillar ATPase activity was affected by test temperature (P<0.001). Like swimming speed, enzyme activity was greater in the cool-reared tadpoles than in the warm-reared tadpoles, a difference that was significant when assayed at 15 degrees C (P<0. 01). These results suggest a mechanism for developmental temperature effects on locomotor performance observed in other taxa.     

Seasonal effects on oocyte developmental competence in sows experiencing pregnancy loss

Recently, oocyte quality in sows culled for reasons unrelated to fertility was found to decline during the period of seasonal infertility. Wean-to-service interval (WSI) has also been associated with pregnancy loss in sows mated during the period of seasonal infertility. The aims of this study were to determine whether WSI and season are associated with changes in oocyte developmental competence in sows experiencing early (before Day 35 of gestation) and late (after Day 35 of gestation) pregnancy loss. Ovaries were collected in pairs from sows sourced from commercial piggeries that were culled for reasons related to infertility after being mated in summer and winter/spring. Sows were grouped according to their pregnancy loss type, their previous WSI and the presence or absence of corpora lutea (CL) on their ovaries. Oocyte developmental competence was assessed following in vitro maturation, artificial activation and parthenote development in vitro. In sows culled for early-pregnancy loss, there was a greater number of CL present on ovaries collected in spring compared to those collected in summer (11.57±3.3 vs. 9.26±0.99; P<0.05). Also, the proportion of oocytes developing to the blastocyst stage was greater in summer than in spring (55.9±5.2% vs. 31.2±6.4%; P<0.05). In sows culled for late-pregnancy loss, a greater proportion of oocytes developed to the blastocyst stage in winter compared with late-spring (64.3±7.0% vs. 34.1±6.6%; P<0.05). In addition, the blastocyst formation rate of oocytes was lower in sows that displayed a WSI≤6 days than in sows that displayed a WSI>6 days (37.8±7.3% vs. 62.2±6.9%; P<0.05). The results of the present study indicate that sows culled for pregnancy loss exhibit seasonal changes in oocyte developmental competence. The mechanism which causes WSI to be prolonged does not appear to result in reduced oocyte developmental competence. While poor oocyte quality and the mechanism that increases WSI may contribute to pregnancy loss during the seasonal infertility period, the findings suggest that these factors are not the main drivers of early and late pregnancy loss throughout the year.     

The effects of five organophosphorus insecticides on thermal stress in tadpoles of the Pacific tree frog, Hyla regilla

Three week old tadpoles of Hyla regilla were exposed to the insecticides temephos, fenthion, methyl parathion, chlorpyrifos, and malathion for 24 h at concentrations less than and at field applied rates as recommended for mosquito control in California. The thermal tolerance was significantly lowered by all chemicals and concentrations tested. Chlorpyrifos and methyl parathion were the most active compounds tested whereas malathion was the least toxic to H regilla tadpoles. Activity was depressed, in comparison to control tadpoles, by the chemicals methyl parathion (100 ppb) and malathion (500 ppb).     

Isolation and characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas

Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.     

Pool desiccation and developmental thresholds in the common frog, Rana temporaria

The developmental threshold is the minimum size or condition that a developing organism must have reached in order for a life-history transition to occur. Although developmental thresholds have been observed for many organisms, inter-population variation among natural populations has not been examined. Since isolated populations can be subjected to strong divergent selection, population divergence in developmental thresholds can be predicted if environmental conditions favour fast or slow developmental time in different populations. Amphibian metamorphosis is a well-studied life-history transition, and using a common garden approach we compared the development time and the developmental threshold of metamorphosis in four island populations of the common frog Rana temporaria: two populations originating from islands with only temporary breeding pools and two from islands with permanent pools. As predicted, tadpoles from time-constrained temporary pools had a genetically shorter development time than those from permanent pools. Furthermore, the variation in development time among females from temporary pools was low, consistent with the action of selection on rapid development in this environment. However, there were no clear differences in the developmental thresholds between the populations, indicating that the main response to life in a temporary pool is to shorten the development time.     

Where movement happens: scale‐dependent landscape effects on genetic differentiation in the European tree frog

Functional connectivity among fragmented populations depends on the landscape matrix between occupied habitat patches and its effect on the frequency of animal movement and gene flow. The quantification of landscape effects on gene flow should therefore be scale‐dependent. Here, we explored the impact of different spatial scales in a landscape genetic analysis of the European tree frog Hyla arborea in a fragmented landscape in Switzerland. We examined the effects of landscape elements and geographic distance on genetic differentiation at three distance classes reflecting varying frequencies of tree frog movement. We calculated pairwise F_ST‐values and assembled 16 landscape elements within 1 km wide corridors between all pairs of tree frog breeding sites. Per distance class, we computed a multiple regression model with stepwise backward elimination and permutation testing. At distances of<2 km, only a larger river acted as a barrier to gene flow. At distances>2 km, geographic distance had a negative effect on gene flow as had landscape elements such as forests and roads. In general, hedgerows and various structure‐rich landscape elements positively affected gene flow. As we found distinct scale‐dependent landscape effects on gene flow, future landscape genetic studies should analyse the effects of landscape variables at different spatial dimensions relevant for the movement and dispersal of the study organisms. Corresponding studies should also carefully consider relevant correlations among the landscape elements tested and should preferentially replicate their analysis at the landscape‐level in order to avoid idiosyncratic results owing to the particular scale and landscape studied.     

Hormone induced chromatophore changes in the European tree frog, Hyla arborea, in vitro

The colours of the European tree frog, Hvlu urhorea , depend on three types of chromatophores: in dermo-epidermal direction melanophores, iridophores, and xanthophores. The ability ofthis species to assume a wide range ofcolours implies that very extensive changes in the chromatophores take place, which in turn require control by several regulating factors. The responses of the different chromatophore types to hormones with known melanophore-affecting abilities (α-MSH, β-MSH, ACTH, melatonin) were tested in an in vitro system (freshly explanted skin) using reflectance microspectrophotometry, light microscopy and time-lapse cinemicrography.
α-MSH, β-MSH and ACTH all induce a rapid dispersion of melanosomes during the 10 min after addition. The degree of pigment dispersion induced by ACTH is slightly less than after stimulation with α-MSH or β-MSH.
The iridophores react to MSH or ACTH treatment with a contraction of the entire cell (causing a reduction in reflecting area), and a change in orientation of the platelets, causing a decrease in selective reflectance. The iridophores appear to be especially sensitive to ACTH. A very striking feature of the iridophores when studied with time-lapse cinematography is their strong pulsations (approx. once per minute).
The xanthophores react to MSH and ACTH with a contraction. These cells appear to be sensitive to β-MSH in particular.
Melatonin strongly counteracts the effects of α-MSH, β-MSH and ACTH on all chromatophores.
These studies confirm the dynamic nature not only of the melanophores, but also of the iridophores and xanthophores, as pointed out by Schmidt (1920) and Nielsen (1978a). Furthermore the differences in the time course of the stimulation of the different types of chromatophores by various hormones may provide an experimental basis for the explanation of colour changes in Hyfa arboreu.     

Environmental factors influencing trematode prevalence in grey tree frog (Hyla versicolor) tadpoles in southern Ontario

The emergence or increased prevalence of various parasites may be linked to alterations in host-parasite interactions caused by environmental changes. We investigated prevalence of trematode infections in grey tree frog (Hyla versicolor) tadpoles from ponds in nonagricultural settings versus ponds adjacent to cornfields in southern Ontario. We found that agricultural activity was a significant factor in determining the percentage of tadpoles infected by 1 or more trematodes from 1 or more species (combined trematode infection). However, we found no associations between combined trematode infection and forest cover; pond size; road density; and measures of water quality, such as nitrate level and the presence of the herbicide atrazine. Although combined trematode prevalence was associated only with agricultural activity, prevalence of Alaria species showed a positive association with forest cover. This latter result probably reflects the importance of habitat suitability for the canid definitive hosts of this trematode species.     

Neuroepithelial bodies in the lung of the tree frog,Hyla arborea L.

Summary The endocrine-like cells (ELC), which together with nerve endings form the neuroepithelial bodies, are located on primary and secondary septa in the non-ciliated epithelium of the lung of Hyla arborea. ELC protrude markedly toward the lumen of the lung and are surrounded by pneumocytes, which separate ELC from the lumen by thin cytoplasmic processes. ELC possess a light cytoplasm containing two types of granules: (i) numerous small granules, 50–110 nm in diameter, and (ii) single large granules, 290–860 nm in diameter. Numerous nerve fibers, often forming synaptic junctions, can be observed in contact with ELC.This study was supported by a grant No. 476/II from the Polish Academy of Sciences     

The effects of aluminum and acid on the gill morphology in rainbow trout,Salmo gairdneii

Synopsis The objective was to determine the effects of acid and aluminum in acidified hard and soft water on the histology and morphometry of rainbow trout gills, and to determine relevant toxicity indicators within the gill tissue. Acid and aluminum promoted measurable primary epithelial hyperplasia which proved to be a reliable biological indicator of acid and aluminum contamination and possibly of some predictive value. Low levels of aluminum and acid resulted in hypertrophied chloride cells, suggesting a role in adapting to the contaminants. High concentrations of aluminum (>10 molI^-1) caused chloride cell necrosis and consequently a decline in cell numbers over time. Aluminum precipitates accumulating within the chloride cell cytoplasm probably lead to impaired function prior to cell degeneration. The morphological alterations resulted in a decrease in water space between secondary lamellae (up to 40% within 14 d) which may reduce the efficiency of gas exchange. Twice the aluminum was required in hard water to elicit a similar soft water tissue response. Pathological changes were more severe with aluminum at pH 5.2 than at pH 4.7; results of aluminum speciation suggest that both labile and non-labile fractions are responsible for the induction of gill lesions. Low levels of aluminum may protect fish from the effects of high hydrogen ion concentration.