Activity of Sphaerotheca breviceps tadpoles in response to chemical cues of the predaceous tadpoles Hoplobatrachus tigerinus

Tadpoles of Sphaerotheca breviceps raised in the laboratory from the egg stage, and hence lacking prior experience of a predator or its odors, were tested to examine their responses to a predator’s (tadpoles of Hoplobatrachus tigerinus) water-borne chemical cues. The stimulus solution was obtained following 24 h of rearing tadpoles of H. tigerinus (one tadpole per 200 mL water) that were not fed during this period. Upon exposure to the stimulus solution the activity of S. breviceps tadpoles decreased by about 90% within 5 min. Their resting period increased significantly over baseline activity, whereas the swimming period, distance traversed, and swimming spurts declined. However, whenever a test tadpole moved, its swimming velocity was high in response to stimulus solution. The antipredatory responses declined with increase in time of storage of the stimulus solution, indicating decay of the predator’s chemical cues. The findings suggest that (1) antipredator defense strategies of S. breviceps do not require prior experience of predators, (2) the predator’s chemical cues are labile in nature, and (3) the response of prey tadpoles to such cues is similar to reported behavior of anuran tadpoles in response to real predators and alarm cues.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Indirect effects of introduced trout on Cascades frogs (Rana cascadae) via shared aquatic prey

1. The introduction of trout to montane lakes has negatively affected amphibian populations across the western United States. In northern California’s Klamath–Siskiyou Mountains, introduced trout have diminished the distribution and abundance of a native ranid frog, Rana (=Lithobates) cascadae. This is primarily thought to be the result of predation on frog larvae. However, if trout feed on larval aquatic insects that are available to R. cascadae only after emergence, then resource competition may also affect this declining native amphibian. 2. Stomach contents of R. cascadae were compared between lakes that contained trout and those from which introduced trout were removed. Total prey mass in stomach contents relative to frog body mass was not significantly different between lakes with fish and fish‐removal lakes, but in the former R. cascadae consumed a smaller proportion of adult aquatic insects. The stomach contents of fish included larvae of aquatic insects that are, as adults, eaten by R. cascadae. 3. Rana cascadae consumed fewer caddisflies (Trichoptera) and more grasshoppers (Orthoptera) at lakes with higher densities of fish. At lakes with greater aquatic habitat complexity, R. cascadae consumed more water striders (Hemiptera: Gerridae) and terrestrial spiders (Araneae). 4. We suggest that reductions in the availability of emerging aquatic insects cause R. cascadae to consume more terrestrial prey where trout are present. Thus, introduced trout may influence native amphibians directly through predation and, indirectly, through pre‐emptive resource competition.     

Phylogeographic inference using Bayesian model comparison across a fragmented chorus frog species complex

Fragmented species complexes provide an interesting system for investigating biogeographic history and the present distribution of genetic variation. Recent advances in sequencing technology and statistical phylogeography enable the collection and rigorous analysis of large multilocus data sets, but designing studies that produce meaningful phylogeographic inferences remains challenging. We implemented a Bayesian model comparison approach to investigate previous biogeographic hypotheses while simultaneously inferring the presence of genetic structure in a chorus frog species complex. The Illinois chorus frog (Pseudacris illinoensis), originally described as a subspecies of the broadly distributed Strecker's chorus frog (Pseudacris streckeri), occurs in small, disjunct regions associated with scarce sand prairie habitats that have been impacted by human development. We used high‐throughput sequencing to develop and collect a multitiered genetic data set comprised of three different marker types (23 anonymous nuclear sequence loci, four mitochondrial genes and 14 microsatellite loci) designed to address questions across different evolutionary timescales. Phylogenetic analyses uncovered a deep divergence between populations in the Edwards Plateau of central Texas and all other P. streckeri/P. illinoensis populations, but suggest the disjunct distribution of P. illinoensis occurred more recently. Our best‐supported migration model is consistent with the hypothesis that central Texas represented a refugium from which populations expanded via multiple routes. This model also indicates that disjunct northern and southern regions of P. illinoensis should be considered genetically distinct management units. Our study provides an evolutionary context for future studies and conservation efforts in P. illinoensis and demonstrates the utility of model‐based approaches for phylogeographic inference.     

Highly polymorphic microsatellite markers for the highly polymorphic strawberry poison-dart frog and some of its congeners

Members of the poison-dart frog genus Oophaga, including the strawberry poison-dart frog (O. pumilio) display among the most striking examples of color polymorphism of any amphibians. We developed twelve novel microsatellite markers with di-, tri-, and tetra-nucleotide repeats for this genus. These loci are highly polymorphic with between 2 and 29 alleles (average = 14.7) and high heterozygosity (H _O = 0.704). These highly polymorphic markers should be useful for resolving fine-scale genetic differences between the different color morphs of these highly variable species, for investigations into the ecological importance of this variation, and for determining the effects of habitat loss and fragmentation on population persistence in these species.     

In a rough spot: Declines in Arthroleptella rugosa calling densities are explained by invasive pine trees

Basing conservation interventions on evidence is important for justifying their associated cost and gauging their effectiveness. For amphibians, the number of studies available to support conservation action plans is limited. Here, we sought to determine the effect of invasive pine trees on the calling densities of a Critically Endangered frog species, endemic to the Western Cape province in South Africa. The Rough Moss frog, Arthroleptella rugosa, is an anuran restricted to a small patch of fire-driven fynbos habitat prone to invasion by Cluster Pines, Pinus pinaster. We use acoustic spatially explicit capture–recapture methods to estimate frog densities at multiple sites (n = 12) over ten years. Sites were classified as invaded or uninvaded by P. pinaster, and this information, along with the time since the last fire, were used as explanatory variables for frog density in a generalized linear mixed model. Frog densities were found to be significantly affected by P. pinaster invasion status. At invaded sites, there was a negative relationship between call densities and time since fire, while at uninvaded sites the same relationship was positive. These results confirm previous suggestions that invasive pine trees cause population declines in A. rugosa. Our findings can be used to support conservation interventions for A. rugosa, specifically the use of fire in an adaptive management context to control pine invasion across its range.     

Remobilization of <Emphasis Type="Italic">Tol2</Emphasis> transposons in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Background  

The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.     

Prevalence of endoparasitic mites on four West African leaf‐litter frogs depends on habitat humidity

Amphibian species are known to carry endoparasitic mites. The infestation probability, prevalence, and intensity of mites vary among species and habitats. Mites of the genus Endotrombicula are known to infest African and Malagasy frogs. However, the factors leading to an increase in the probability of mite infestation are unknown. To test for inter‐ and intraspecific differences in infestation probability and its potential correlation with sex, age, habitat preferences, and/or season within a species‐rich West African leaf‐litter frog assemblage, we examined more than 6,800 individual frogs for the presence of mites throughout two independent time increments, 1999–2000 and 2016–2017. We found only members of the leaf‐litter frog genus Phyrnobatrachus to be infested, while other syntopically occurring genera were not affected. Within Phrynobatrachus, only four out of eight species were infested. Mites prevalence differed between species (highest P. phyllophilus, followed by P. alleni), sex (males higher than females in P. alleni and P. phyllophilus), and age (adults higher than juveniles in P. alleni), as well as season (more mites during wet than dry season in P. phyllophilus). The prevalence of mite infestation did not influence mate choice in P. alleni. Increased humidity showed a clear positive effect on infestation prevalence. We also detected a marked decrease in the prevalence of mites from 1999–2000 to 2016–2017, a period during which climatological changes within the study area have been reported with a tendency toward drier conditions. The decrease in mite infestation prevalence over time might be a signal of increasingly drier conditions.     

Urban growth threatens the lowland Amazonian Manaus harlequin frog which represents an evolutionarily significant unit within the genus Atelopus (Amphibia: Anura: Bufonidae)

The Manaus harlequin frog is an evolutionarily significant clade within the Atelopus hoogmoedi species complex. Analyses of 16S and COI concatenated sequences support Atelopus from the Manaus region as an evolutionary significant unit, sister of all species of a Guiana Shield clade. A previous study showed that subtle changes in stream characteristics influence the Manaus harlequin frog occurrence and density variation at local-scale in a reserve on the outskirts of Manaus. As deforestation is approaching areas where the Manaus harlequin frog occurs, we asked how site and landscape heterogeneity influence the geographic boundaries, occurrence patterns, and density variation of the Manaus harlequin frog. We searched for the frog in 80 plots that measured 250 m by 4 m on banks of first- to third-order streams during the rainy seasons in 2012–2013 and 2016–2019. The plot distribution covered all likely areas of occurrence of the Manaus harlequin frog and extended to the areas where it is substituted by its geographically closest relative on the Guiana Shield. Ecological drivers related to climate, flooding events, and forest structure apparently restrict the Manaus harlequin frog to a patchy distribution in a narrow portion of the interfluve between the Negro and Uatuma Rivers. Densities of individuals varied in response to subtle changes in floodplain and stream characteristics. The Manaus harlequin frog is associated with a very specific habitat that is directly affected by the growth of Manaus, the largest city in Amazonia. We conclude that it is endangered and urgent actions are required for its conservation.     

Voltage-dependent calcium currents in cultured frog skeletal myocytes

Voltage-dependent calcium currents (I _Ca) in cultured embryonic skeletal frog myocytes at the stages of development ranging from one to six days were studied using the whole-cell patch-clamp recording technique. Fast dihydropyridine (DHP)-nonsensitiveI _Ca and slow DHP-sensitiveI _Ca were recorded during the early stages of myogenesis. These currents do not differ in their kinetics from theI _Ca found in the adult frog muscle. Two slow voltage-dependent DHP-sensitiveI _Ca were found. In terms of its activation time, the first slow I_Ca is similar toI _Ca described in phasic skeletal muscle fibers. The second slowI _Ca observed in about 10% of cells examined is similar toI _Ca described in tonic skeletal muscle fibers of adult frog. In some cases, two slow currents with different kinetics were recorded in the same myocyte.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 44–48, January–February, 1994.     

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.     

Foraging traits of native predators determine their vulnerability to a toxic alien prey

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Purification and Characterization of Albumin from Frog Skin of <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis>

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K _i values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.     

Regional variation in drivers of connectivity for two frog species (Rana pretiosa and R. luteiventris) from the U.S. Pacific Northwest

Comparative landscape genetics has uncovered high levels of variability in which landscape factors affect connectivity among species and regions. However, the relative importance of species traits versus environmental variation for predicting landscape patterns of connectivity is unresolved. We provide evidence from a landscape genetics study of two sister taxa of frogs, the Oregon spotted frog (Rana pretiosa) and the Columbia spotted frog (Rana luteiventris) in Oregon and Idaho, USA. Rana pretiosa is relatively more dependent on moisture for dispersal than R. luteiventris, so if species traits influence connectivity, we predicted that connectivity among R. pretiosa populations would be more positively associated with moisture than R. luteiventris. However, if environmental differences are important drivers of gene flow, we predicted that connectivity would be more positively related to moisture in arid regions. We tested these predictions using eight microsatellite loci and gravity models in two R. pretiosa regions and four R. luteiventris regions (n = 1,168 frogs). In R. pretiosa, but not R. luteiventris, connectivity was positively related to mean annual precipitation, supporting our first prediction. In contrast, connectivity was not more positively related to moisture in more arid regions. Various temperature metrics were important predictors for both species and in all regions, but the directionality of their effects varied. Therefore, the pattern of variation in drivers of connectivity was consistent with predictions based on species traits rather than on environmental variation.     

The potential impact of simulated ground-water withdrawals on the oviposition,larval development,and metamorphosis of pond-breeding frogs

Wetland hydroperiod is a key factor for the reproductive success of pond-breeding amphibians. Ground-water withdrawals may cause intermittent ponds to dry prematurely, potentially affecting amphibian development. In three intermittent ponds, we monitored hydrology and tracked oviposition, larval development, and metamorphosis for three frog species that represented a range of breeding phenologies. The three species were the southern leopard frog (Lithobates sphenocephalus), spring peeper (Pseudacris crucifer), and Pine Barrens treefrog (Hyla andersonii). We simulated ground-water withdrawals by subtracting from 5 to 50 cm (in 5-cm increments) from the measured water-depth values at the ponds over a short-term (2-year) period and a long-term (10-year) period to estimate the potential impact of hydroperiod alterations on frog development. Short-term simulations indicated that 5 and 10 cm water-depth reductions would have resulted in little or no impact to hydroperiod or larval development and metamorphosis of any of the species. Noticeable impacts were estimated to occur for reductions ≥15 cm. Long-term simulations showed that impacts to the appearance of the first pre-metamorphs and metamorphs would have occurred at reductions ≥10 cm and impacts to initial egg deposition would have occurred at reductions ≥20 cm. For all simulations, successively greater reductions would have caused increasing impacts that varied by species and pond, with the 50-cm reductions shortening hydroperiods enough to practically eliminate the possibility of larval development and metamorphosis for all three species. Compared to the spring peeper and southern leopard frog, the estimated impacts of the simulations on the various life stages were the greatest for the Pine Barrens treefrog.     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Links between prey assemblages and poison frog toxins: A landscape ecology approach to assess how biotic interactions affect species phenotypes

Ecological studies of species pairs showed that biotic interactions promote phenotypic change and eco‐evolutionary feedbacks. However, it is unclear how phenotypes respond to synergistic interactions with multiple taxa. We investigate whether interactions with multiple prey species explain spatially structured variation in the skin toxins of the neotropical poison frog Oophaga pumilio. Specifically, we assess how dissimilarity (i.e., beta diversity) of alkaloid‐bearing arthropod prey assemblages (68 ant species) and evolutionary divergence between frog populations (from a neutral genetic marker) contribute to frog poison dissimilarity (toxin profiles composed of 230 different lipophilic alkaloids sampled from 934 frogs at 46 sites). We find that models that incorporate spatial turnover in the composition of ant assemblages explain part of the frog alkaloid variation, and we infer unique alkaloid combinations across the range of O. pumilio. Moreover, we find that alkaloid variation increases weakly with the evolutionary divergence between frog populations. Our results pose two hypotheses: First, the distribution of only a few prey species may explain most of the geographic variation in poison frog alkaloids; second, different codistributed prey species may be redundant alkaloid sources. The analytical framework proposed here can be extended to other multitrophic systems, coevolutionary mosaics, microbial assemblages, and ecosystem services.     

Endothelial retinoblastoma protein reduces abdominal aortic aneurysm development via promoting DHFR/NO pathway-mediated vasoprotection

Carbamoyl phosphate synthetase I (CPS1) represents an important regulatory enzyme of the urea cycle that mediates the ATP-driven reaction ligating ammonium, carbonate, and phosphate to form carbamoyl phosphate. The freeze-tolerant wood frog (Rana sylvatica or Lithobates sylvaticus) accumulates high concentrations of urea during bouts of freezing to detoxify any ammonia generated and to contribute as a cryoprotectant thereby helping to avoid freeze damage to cells. Purification of CPS1 to homogeneity from wood frog liver was performed in control and frozen wood frogs by a three-step chromatographic process. The affinity of CPS1 for its three substrates was tested in the purified control and freeze-exposed enzyme under a variety of conditions including the presence and absence of the natural cryoprotectants urea and glucose. The results demonstrated that affinity for ammonium was higher in the freeze-exposed CPS1 (1.26-fold) and that with the addition of 400 mM glucose it displayed higher affinity for ATP (1.30-fold) and the obligate activator N-acetylglutamate (1.24-fold). Denaturation studies demonstrated the freeze-exposed enzyme was less thermally stable than the control with an unfolding temperature approximately 1.5 °C lower (52.9 °C for frozen and 54.4 °C for control). The control form of CPS1 had a significantly higher degree of glutarylated lysine residues (1.42-fold increase) relative to the frozen. The results suggest that CPS1 activation and maintenance of urea cycle activity despite the hypometabolic conditions associated with freezing are important aspects in the metabolic survival strategies of the wood frog.

     

Phylogeography of the poison frog Mantella viridis (Amphibia: Mantellidae) reveals chromatic and genetic differentiation across ecotones in northern Madagascar

Mantella viridis is a threatened poison frog species endemic to the ecologically very heterogeneous northern region of Madagascar. The existence of several colour morphs within M. viridis and its very low genetic differentiation to the allopatrically distributed Mantella ebenaui raise questions about the processes driving the differentiation between these poison frog populations and about their taxonomic status. Using a DNA fragment of 476 nucleotides of the mitochondrial cytochrome b gene from 240 individuals of this species complex, we investigated the genetic variability of all known colour morphs of M. viridis, sampling this species throughout its known range, as well as several populations of M. ebenaui. Our genetic results confirm that M. viridis and M. ebenaui are closely related but reveal that no haplotype sharing occurs between these two taxa. Further, our molecular analyses provided evidence for barriers to gene flow among some of the colour morphs. Estimates of overlap of bioclimatic envelopes as assessed by ecological niche modelling also suggest a distinct bioclimatic niche of some of the lineages studied.     

Cytotoxic peptides with insulin‐releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae)

Peptidomic analysis of norepinephrine‐stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host‐defense peptides differing by a single amino acid residue belonging to the brevinin‐1 family (brevinin‐1ITa and ‐1ITb), a peptide belonging to the temporin family (temporin‐ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine‐sulphoxide forms of brevinin‐1ITa and ‐1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin‐1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non‐small cell lung adenocarcinoma A549 cells (LC₅₀ = 18 μM), breast adenocarcinoma MDA‐MB‐231 cells (LC₅₀ = 8 μM) and colorectal adenocarcinoma HT‐29 cells (LC₅₀ = 18 μM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC₅₀ = 7 μM). Temporin‐ITa (VFLGAIAQALTSLLGKL.NH₂) was between three and fivefold less potent against these cells. Brevinin‐1ITa inhibited growth of both Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin‐ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN‐BD11 clonal β‐cells at concentrations ≥1 nM, but brevinin‐1ITa was cytotoxic to the cells at concentrations ≥3 μM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.