Insights into caudate amphibian skin secretions with a focus on the chemistry and bioactivity of derived peptides.

Amphibians are well-known for their ability to produce and secrete a mixture of bioactive substances in specialized skin glands for the purpose of antibiotic self-protection and defense against predators. Some of these secretions contain various small molecules, such as the highly toxic batrachotoxin, tetrodotoxin, and samandarine. For some time, the presence of peptides in amphibian skin secretions has attracted researchers, consisting of a diverse collection of - to the current state of knowledge - three to 104 amino acid long sequences. From these more than 2000 peptides many are known to exert antimicrobial effects. In addition, there are some reports on amphibian skin peptides that can promote wound healing, regulate immunoreactions, and may serve as antiparasitic and antioxidative substances. So far, the focus has mainly been on skin peptides from frogs and toads (Anura), eclipsing the research on skin peptides of the ca. 700 salamanders and newts (Caudata). Just recently, several novel observations dealing with caudate peptides and their structure-function relationships were reported. This review focuses on the chemistry and bioactivity of caudate amphibian skin peptides and their potential as novel agents for clinical applications.

Successful Drug-Mediated Host Clearance of Batrachochytrium salamandrivorans.

Skin fungi are among the most dangerous drivers of global amphibian declines, and few mitigation strategies are known. For Batrachochytrium salamandrivorans (Chytridiomycota), available treatments rely on temperature, partially combined with antifungal drugs. We report the clearance of B. salamandrivorans in 2 urodelan species using a solely drug-based approach.

Phylogenetic relationships and systematics of the Amazonian poison frog genus Ameerega using ultraconserved genomic elements.

The Amazonian poison frog genus Ameerega is one of the largest yet most understudied of the brightly colored genera in the anuran family Dendrobatidae, with 30 described species ranging throughout tropical South America. Phylogenetic analyses of Ameerega are highly discordant, lacking consistency due to variation in data types and methods, and often with limited coverage of species diversity in the genus. Here, we present a comprehensive phylogenomic reconstruction of Ameerega, utilizing state-of-the-art sequence capture techniques and phylogenetic methods. We sequenced thousands of ultraconserved elements from over 100 tissue samples, representing almost every described Ameerega species, as well as undescribed cryptic diversity. We generated topologies using maximum likelihood and coalescent methods and compared the use of maximum likelihood and Bayesian methods for estimating divergence times. Our phylogenetic inference diverged strongly from those of previous studies, and we recommend steps to bring Ameerega taxonomy in line with the new phylogeny. We place several species in a phylogeny for the first time, as well as provide evidence for six potential candidate species. We estimate that Ameerega experienced a rapid radiation approximately 7-11 million years ago and that the ancestor of all Ameerega was likely an aposematic, montane species. This study underscores the utility of phylogenomic data in improving our understanding of the phylogeny of understudied clades and making novel inferences about their evolution.

Sexual Dichromatism Drives Diversification within a Major Radiation of African Amphibians.

Theory predicts that sexually dimorphic traits under strong sexual selection, particularly those involved with intersexual signaling, can accelerate speciation and produce bursts of diversification. Sexual dichromatism (sexual dimorphism in color) is widely used as a proxy for sexual selection and is associated with rapid diversification in several animal groups, yet studies using phylogenetic comparative methods to explicitly test for an association between sexual dichromatism and diversification have produced conflicting results. Sexual dichromatism is rare in frogs, but it is both striking and prevalent in African reed frogs, a major component of the diverse frog radiation termed Afrobatrachia. In contrast to most other vertebrates, reed frogs display female-biased dichromatism in which females undergo color transformation, often resulting in more ornate coloration in females than in males. We produce a robust phylogeny of Afrobatrachia to investigate the evolutionary origins of sexual dichromatism in this radiation and examine whether the presence of dichromatism is associated with increased rates of net diversification. We find that sexual dichromatism evolved once within hyperoliids and was followed by numerous independent reversals to monochromatism. We detect significant diversification rate heterogeneity in Afrobatrachia and find that sexually dichromatic lineages have double the average net diversification rate of monochromatic lineages. By conducting trait simulations on our empirical phylogeny, we demonstrate that our inference of trait-dependent diversification is robust. Although sexual dichromatism in hyperoliid frogs is linked to their rapid diversification and supports macroevolutionary predictions of speciation by sexual selection, the function of dichromatism in reed frogs remains unclear. We propose that reed frogs are a compelling system for studying the roles of natural and sexual selection on the evolution of sexual dichromatism across micro- and macroevolutionary timescales.

Idiosyncratic responses to climate-driven forest fragmentation and marine incursions in reed frogs from Central Africa and the Gulf of Guinea Islands.

Organismal traits interact with environmental variation to mediate how species respond to shared landscapes. Thus, differences in traits related to dispersal ability or physiological tolerance may result in phylogeographic discordance among co-distributed taxa, even when they are responding to common barriers. We quantified climatic suitability and stability, and phylogeographic divergence within three reed frog species complexes across the Guineo-Congolian forests and Gulf of Guinea archipelago of Central Africa to investigate how they responded to a shared climatic and geological history. Our species-specific estimates of climatic suitability through time are consistent with temporal and spatial heterogeneity in diversification among the species complexes, indicating that differences in ecological breadth may partly explain these idiosyncratic patterns. Likewise, we demonstrated that fluctuating sea levels periodically exposed a land bridge connecting Bioko Island with the mainland Guineo-Congolian forest and that habitats across the exposed land bridge likely enabled dispersal in some species, but not in others. We did not find evidence that rivers are biogeographic barriers across any of the species complexes. Despite marked differences in the geographic extent of stable climates and temporal estimates of divergence among the species complexes, we recovered a shared pattern of intermittent climatic suitability with recent population connectivity and demographic expansion across the Congo Basin. This pattern supports the hypothesis that genetic exchange across the Congo Basin during humid periods, followed by vicariance during arid periods, has shaped regional diversity. Finally, we identified many distinct lineages among our focal taxa, some of which may reflect incipient or unrecognized species.

Leapfrogging into new territory: How Mascarene ridged frogs diversified across Africa and Madagascar to maintain their ecological niche.

The Mascarene ridged frog, Ptychadena mascareniensis, is a species complex that includes numerous lineages occurring mostly in humid savannas and open forests of mainland Africa, Madagascar, the Seychelles, and the Mascarene Islands. Sampling across this broad distribution presents an opportunity to examine the genetic differentiation within this complex and to investigate how the evolution of bioclimatic niches may have shaped current biogeographic patterns. Using model-based phylogenetic methods and molecular-clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the group based on mitochondrial 16S rRNA and cytochrome b (cytb) genes and the nuclear RAG1 gene from 173 individuals. Haplotype networks were reconstructed and species boundaries were investigated using three species-delimitation approaches: Bayesian generalized mixed Yule-coalescent model (bGMYC), the Poisson Tree Process model (PTP) and a cluster algorithm (SpeciesIdentifier). Estimates of similarity in bioclimatic niche were calculated from species-distribution models (maxent) and multivariate statistics (Principal Component Analysis, Discriminant Function Analysis). Ancestral-area reconstructions were performed on the phylogeny using probabilistic approaches implemented in BioGeoBEARS. We detected high levels of genetic differentiation yielding ten distinct lineages or operational taxonomic units, and Central Africa was found to be a diversity hotspot for these frogs. Most speciation events took place throughout the Miocene, including "out-of-Africa" overseas dispersal events to Madagascar in the East and to São Tomé in the West. Bioclimatic niche was remarkably well conserved, with most species tolerating similar temperature and rainfall conditions common to the Central African region. The P. mascareniensis complex provides insights into how bioclimatic niche shaped the current biogeographic patterns with niche conservatism being exhibited by the Central African radiation and niche divergence shaping populations in West Africa and Madagascar. Central Africa, including the Albertine Rift region, has been an important center of diversification for this species complex.

Expanding Distribution of Lethal Amphibian Fungus Batrachochytrium salamandrivorans in Europe.

Emerging fungal diseases can drive amphibian species to local extinction. During 2010-2016, we examined 1,921 urodeles in 3 European countries. Presence of the chytrid fungus Batrachochytrium salamandrivorans at new locations and in urodeles of different species expands the known geographic and host range of the fungus and underpins its imminent threat to biodiversity.

Acute Toxic Effects of the Herbicide Formulation Focus(®) Ultra on Embryos and Larvae of the Moroccan Painted Frog, Discoglossus scovazzi.

For regulatory and scientific purposes, there is a need to understand the sensitivity of a wider variety of wild species of amphibians and the sensitivities within their life stages to chemicals of widespread use such as herbicides. We investigated the acute toxic effects of the herbicide formulation Focus Ultra [with the active ingredient (a.i.) cycloxydim plus solvent naphtha and sodium dioctylsulphosuccinate as added substances] on embryos and early stage larvae of the Moroccan painted frog (Discoglossus scovazzi). Different clinical signs (twitching, convulsion, and narcosis) occurred at 40 and 80 mg/L in embryos (4 and 8 mg a.i./L) and narcotic effects (total immobilization or irregular escape responses) at 10, 15, and 20 mg/L in larvae (1, 1.5, and 2 mg a.i./L). Growth inhibition (total length), starting at 20 mg/L in embryos and 2.5 mg/L in larvae (2 and 0.25 mg a.i./L, respectively) was understood as sign of toxicity (retardation) and not as sign of teratogenicity. However, the connection to teratogenesis remained unclear though total length reduction occurred at concentrations <20 % of the 96-h LC50 value and at a minimum concentration that inhibits growth of only 17 % of the 96-h LC50 value. Starting at 20 mg/L, mortality in embryos significantly increased and at 15 mg/L in early larvae (2 and 1.5 mg a.i./L, respectively). Mortality of larvae was enhanced during the first 24 h of exposure to 15 and 20 mg/L (1.5 and 2 mg a.i./L). Morphology of the embryos remained unobtrusive. In contrary, axial malformations significantly increased in the early larvae starting at 10 mg/L (1 mg a.i./L), a concentration free of lethal effects. In all considered end points, larvae were significantly more sensitive than embryos, probably because of developmental and physiological properties or different exposure and bioavailability of the compound. Focus Ultra induced comparable lethal and immobilization effects in D. scovazzi as it does to standard test organisms in pesticide approval. However, to validate the apparent safety in the field, which is based on calculated surface water concentrations of the a.i., more data on real contamination levels is necessary (e.g., peak concentrations, concentrations of added substances). Furthermore, sufficient buffer strips between the farmland and amphibian ponds must be considered, and the effects of the substance on terrestrial life stages have not been assessed yet.

Acute toxic effects of the herbicide formulation and the active ingredient used in cycloxydim-tolerant maize cultivation on embryos and larvae of the African clawed frog, Xenopus laevis.

Most genetically engineered herbicide-tolerant crops are still awaiting approval in Europe. There is, however, a recent trend for the cultivation of cycloxydim-tolerant maize hybrids for use in maize production. We studied the acute toxic effects of the complementary herbicide Focus(®) Ultra and its active ingredient cycloxydim on embryos and early-stage larvae of the African clawed frog (Xenopus laevis). The results indicate that the herbicide formulation is significantly more toxic than the active ingredient alone. Therefore, it is suggested that the added substances either solely or in a synergistic action with the active ingredient are responsible for adverse effects. The formulation was found to be moderately toxic to embryos but highly toxic to early larvae. Based on calculated teratogenic indices, both cycloxydim and Focus(®) Ultra seem to be non-teratogenic and also the minimum Focus(®) Ultra concentration to inhibit growth in embryos and larvae was close to the LC50 values. The data suggest that tests with the rainbow trout are not in all cases appropriate to assess the risk in aquatically developing anurans. This is demonstrated by 96-h LC50 values, which are for rainbow trout more than 50- to 20-fold higher than for early X. laevis larvae. However, based on worst-case predicted environmental concentrations for surface waters, there is apparently a large safety margin in field use of Focus(®) Ultra if buffer strips between the farm land and the amphibian habitats are regarded.

Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

A danger foreseen is a danger avoided: how chemical cues of different tadpoles influence parental decisions of a Neotropical poison frog.

The protection of offspring against predators and competitors is especially important in organisms using spatially separated breeding resources, impeding the offspring's chances to escape. One example of such isolated reproductive resources are phytotelmata (small water bodies in plant axils), exploited by the Neotropical poison frog Ranitomeya variabilis (Dendrobatidae) for both clutch and tadpole deposition. Because poison frog tadpoles are often cannibalistic, parents tend to avoid deposition with conspecifics. Previous studies have shown that this avoidance is based on chemical cues produced by conspecific tadpoles. Further, cues produced by phylogenetically less-related tadpoles (Bufonidae) were avoided for clutch but not tadpole depositions. We analyzed how the different responses to tadpole cues are triggered. We tested the reactions of parental R. variabilis to tadpole cues of species differing in two aspects: whether or not they are dendrobatids, and whether or not they reproduce in phytotelmata. We found that for clutch deposition, tadpole cues were always avoided, i.e., all tadpoles were treated by the frogs as if they pose a danger to the eggs. However, responses varied for tadpole depositions: while dendrobatid larvae living in phytotelmata were avoided, those breeding in streams were not. Non-poison frog tadpoles were ignored when associated with habitat other than phytotelmata, but they were preferred when living in phytotelmata. This suggests that both phylogeny and tadpole habitat are important triggers for the decisions made by R. variabilis. Only tadpoles using the same breeding resources are considered as relevant for the frog's own larvae (i.e., as a potential danger or food resource), while further decisions are related to evolutionary relationship.

Alpine salamanders at risk? The current status of an emerging fungal pathogen.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the 'salamander plague' ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

Effects of water contamination on site selection by amphibians: experiences from an arena approach with European frogs and newts.

Pesticide residues in breeding ponds can cause avoidance by at least some amphibian species. So far, outdoor experiments have been performed only with artificial pools in areas where the focus species usually occur and new colonization has been observed. Results of this kind of study are potentially influenced by natural disturbances and therefore are of limited comparability. We used an easily manufactured and standardizable arena approach, in which animals in reproductive condition for some hours had a choice among pools with different concentrations of a contaminant. Because there has been much debate on the potential environmental impacts of glyphosate-based herbicides, we investigated the impact of glyphosate isopropylamine salt (GLY-IS), Roundup LB PLUS (RU-LB-PLUS), and glyphosate's main metabolite aminomethylphosphonic acid (AMPA) on individual residence time in water. The following European amphibian species were tested: Common frog (Rana temporaria), Palmate newt (Lissotriton helveticus), and Alpine newt (Ichthyosaura alpestris). The residence time in water was not significantly affected by concentrations below or slightly above the European Environmental Quality Standards for AMPA or the German "worst-case" expected environmental concentrations for GLY-IS and RU-LB-PLUS. Occasionally, microclimatic cofactors (nightly minimum ground temperature, water temperature) apparently influenced the residence time. The major drawback of such quick behavior studies is that results can only be transferred to perception and avoidance of contaminated water but not easily to site selection by amphibians. For example, testing oviposition site selection requires more natural water bodies and more time. Hence, to develop a standard procedure in risk assessment, an intermediate design between an arena approach, as presented here, and previously performed field studies should be tested.

COVID-19 infection in CVID patients: What we know so far.

INTRODUCTION: In patients with common variable immunodeficiency (CVID), immunological response is compromised. Knowledge about COVID-19 in CVID patients is sparse. We, here, synthesize current research addressing the level of threat COVID-19 poses to CVID patients and the best-known treatments. METHOD: Review of 14 publications. RESULTS: The number of CVID patients with moderate to severe (~29%) and critical infection courses (~10%), and the number of fatal cases (~13%), are increased compared to the general picture of COVID-19 infection. However, this might be an overestimate. Systematic cohort-wide studies are lacking, and asymptomatic or mild cases among CVID patients occur that can easily remain unnoticed. Regular immunoglobulin replacement therapy was administered in almost all patients, potentially explaining why the numbers of critical and fatal cases were not higher. In addition, the application of convalescent plasma was demonstrated to have positive effects. CONCLUSIONS: COVID-19 poses an elevated threat to CVID patients. However, only systematic studies can provide robust information on the extent of this threat. Regular immunoglobulin replacement therapy is beneficial to combat COVID-19 in CVID patients, and best treatment after infection includes the use of convalescent plasma in addition to common medication.

Morphology of lentic and lotic tadpoles from Madagascar.

BACKGROUND: The morphology of anuran larvae is suggested to differ between species with tadpoles living in standing (lentic) and running (lotic) waters. To explore which character combinations within the general tadpole morphospace are associated with these habitats, we studied categorical and metric larval data of 123 (one third of which from lotic environments) Madagascan anurans. RESULTS: Using univariate and multivariate statistics, we found that certain combinations of fin height, body musculature and eye size prevail either in larvae from lentic or lotic environments. CONCLUSION: Evidence for adaptation to lotic conditions in larvae of Madagascan anurans is presented. While lentic tadpoles typically show narrow to moderate oral discs, small to medium sized eyes, convex or moderately low fins and non-robust tail muscles, tadpoles from lotic environments typically show moderate to broad oral discs, medium to big sized eyes, low fins and a robust tail muscle.

Explanative power of variables used in species distribution modelling: an issue of general model transferability or niche shift in the invasive Greenhouse frog (Eleutherodactylus planirostris).

The use of species distribution models (SDMs) to predict potential distributions of species is steadily increasing. A necessary assumption when projecting models throughout space or time is that climatic niches are conservative, but recent findings of niche shifts during biological invasion of particular plant and animal species have indicated that this assumption is not categorically valid. One reason for observed shifts may relate to variable selection for modelling. In this study, we assess differences in climatic niches in the native and invasive ranges of the Greenhouse frog (Eleutherodactylus planirostris). We analyze which variables are more 'conserved' in comparison to more 'relaxed' variables (i.e. subject to niche shift) and how they influence transferability of SDMs developed with Maxent on the basis of ten bioclimatic layers best describing the climatic requirements of the target species. We focus on degrees of niche similarity and conservatism using Schoener's index and Hellinger distance. Significance of results are tested with null models. Results indicate that the degrees of niche similarity and conservatism vary greatly among the predictive variables. Some shifts can be attributed to active habitat selection, whereas others apparently reflect variation in the availability of climate conditions or biotic interactions between the frogs' native and invasive ranges. Patterns suggesting active habitat selection also vary among variables. Our findings evoke considerable implications on the transferability of SDMs over space and time, which is strongly affected by the choice and number of predictors. The incorporation of 'relaxed' predictors not or only indirectly correlated with biologically meaningful predictors may lead to erroneous predictions when projecting SDMs. We recommend thorough assessments of invasive species' ecology for the identification biologically meaningful predictors facilitating transferability.

Future potential distribution of the emerging amphibian chytrid fungus under anthropogenic climate change.

Anthropogenic climate change poses a major threat to global biodiversity with a potential to alter biological interactions at all spatial scales. Amphibians are the most threatened vertebrates and have been subject to increasing conservation attention over the past decade. A particular concern is the pandemic emergence of the parasitic chytrid fungus Batrachochytrium dendrobatidis, which has been identified as the cause of extremely rapid large-scale declines and species extinctions. Experimental and observational studies have demonstrated that the host-pathogen system is strongly influenced by climatic parameters and thereby potentially affected by climate change. Herein we project a species distribution model of the pathogen onto future climatic scenarios generated by the IPCC to examine their potential implications on the pandemic. Results suggest that predicted anthropogenic climate change may reduce the geographic range of B. dendrobatidis and its potential influence on amphibian biodiversity.

Global Distribution of Common Variable Immunodeficiency (CVID) in the Light of the UNDP Human Development Index (HDI): A Preliminary Perspective of a Rare Disease.

Common variable immunodeficiency (CVID), although the most common primary immunodeficiency in humans, is a rare disease. We explored the spatial global distribution and country-wise prevalence of CVID, based on published data and those available from databases. As a country's medical progress is linked to its technological and socio-economic developmental status, we expected that observed CVID prevalence was linked to human wellbeing. To assess this, we examined the correlation of observed CVID prevalence and the UNDP Human Development Index (HDI), which is a key measure of human development. Seventy-four data sets from 47 countries were available (most of them no older than 10 years). Analyses revealed that observed CVID prevalence ranged from 0.001 to 3.374 per 100,000 (mean 0.676 ± 0.83) and was highest in "high" HDI countries (Spearman's rho = 0.757). Observed prevalence was particularly high in countries where immunodeficiencies are systematically documented in registers. In "low" and "middle" HDI countries, CVID awareness is extremely poor. Assuming that true CVID prevalence does not differ among countries, this study, though preliminary, provides evidence that the discrepancy between observed and (unknown) true prevalence can be clearly linked to the countries' developmental status. As a potential alternative explanation, we briefly discuss the possibility that variation in CVID prevalence is related to human genetic lineage.

Phylogeographic analyses point to long-term survival on the spot in micro-endemic Lycian salamanders.

Lycian salamanders (genus Lyciasalamandra) constitute an exceptional case of micro-endemism of an amphibian species on the Asian Minor mainland. These viviparous salamanders are confined to karstic limestone formations along the southern Anatolian coast and some islands. We here study the genetic differentiation within and among 118 populations of all seven Lyciasalamandra species across the entire genus' distribution. Based on circa 900 base pairs of fragments of the mitochondrial 16SrDNA and ATPase genes, we analysed the spatial haplotype distribution as well as the genetic structure and demographic history of populations. We used 253 geo-referenced populations and CHELSA climate data to infer species distribution models which we projected on climatic conditions of the Last Glacial Maximum (LGM). Within all but one species, distinct phyloclades were identified, which only in parts matched current taxonomy. Most haplotypes (78%) were private to single populations. Sometimes population genetic parameters showed contradicting results, although in several cases they indicated recent population expansion of phyloclades. Climatic suitability of localities currently inhabited by salamanders was significantly lower during the LGM compared to recent climate. All data indicated a strong degree of isolation among Lyciasalamandra populations, even within phyloclades. Given the sometimes high degree of haplotype differentiation between adjacent populations, they must have survived periods of deteriorated climates during the Quaternary on the spot. However, the alternative explanation of male biased dispersal combined with a pronounced female philopatry can only be excluded if independent nuclear data confirm this result.