Midwife Toad Alytes Obstetricans Research Articles

No amphibian chytrid fungus Batrachochytrium dendrobatidis detected in four introduced populations of the midwife toad Alytes obstetricans in eastern England

The amphibian chytrid fungus Batrachochytrium dendrobatidis threatens amphibian species globally as the causative agent of chytridiomycosis, with the introduction of non-native species being one of the pathways that the pathogen can spread to naive populations. We have monitored and screened the common midwife toad Alytes obstetricans in four separate populations in eastern England, to investigate the potential threats to local amphibians. Forty-eight toads across all life stages were swabbed between May 2018 and August 2020, and screened for the presence of B. dendrobatidis DNA using qPCR. None of the samples tested were positive, indicating that it is unlikely that any of the A. obstetricans swabbed were infected with B. dendrobatidis. The populations surveyed represent only a small part of the species range in Britain, consequently a more widespread survey is recommended to increase confidence that British A. obstetricans are free of B. dendrobatidis.

Amphibian loss alters periphyton structure and invertebrate growth in montane streams.

Amphibians are declining worldwide due to a combination of stressors such as climate change, invasive species, habitat loss, pollution and emergent diseases. Although their losses are likely to have important ecological consequences on the structure and functioning of freshwater ecosystems, this issue has been scarcely explored. We conducted an experiment in three montane streams-where primary production is the main source of energy and carbon-to assess the effects of amphibian disappearance (i.e. presence or absence of the common midwife toad Alytes obstetricans, a common species found in pools of these streams) on several aspects of ecosystem functioning and structure: periphyton biomass and chlorophyll a concentration, algal assemblage structure, and growth of macroinvertebrate grazers. We compared four types of experimental enclosures: (i) without macroinvertebrates or amphibians; (ii) with larvae of the caddisfly Allogamus laureatus; (iii) with A. obstetricans tadpoles; and (iv) with both A. laureatus larvae and A. obstetricans tadpoles. The absence of tadpoles increased periphyton biomass, but did not cause differences on inorganic sediment accrual. The algal assemblage had a higher diversity in the absence of tadpoles, and their characteristic taxa differed from the assemblages in presence of tadpoles. A. laureatus presented higher mass in presence of tadpoles; however, tadpole length was not affected by presence of macroinvertebrates. Our results suggest that presence of tadpoles is a driver of periphyton accrual and assemblage structure, acting as top-down control and with key potential consequences on the functioning of montane stream ecosystems.

08. Limb malformations in introduced populations of midwife toad Alytes obstetricans in Great Britain

08. Limb malformations in introduced populations of midwife toad Alytes obstetricans in Great Britain

Microbiome function predicts amphibian chytridiomycosis disease dynamics

BackgroundThe fungal pathogenBatrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood.MethodsMicrobiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection.ResultsMicrobiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes.ConclusionsBd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection.6u4HXENqNsd5f8SBedLBdGVideo

Male common midwife toad Alytes obstetricans depositing eggs in a flowerpot saucer in a suburban garden?

Male common midwife toad Alytes obstetricans depositing eggs in a flowerpot saucer in a suburban garden?

Tadpoles of the midwife toad Alytes obstetricans scavenging carrion

Tadpoles of the midwife toad Alytes obstetricans scavenging carrion

In-situ severe breeding habitat intervention only achieves temporary success in reducing Batrachochytrium dendrobatidis infection

Abstract Chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), is causing sharp declines in amphibian populations around the globe. A substantial research effort has been made to study the disease, including treatments against Bd, but most treatments have been applied to captive amphibians only. We report a study aimed at clearing wild populations of the Common Midwife toad Alytes obstetricans. We removed all larvae from natural breeding sites (cattle troughs) and conducted two types of severe breeding habitat manipulation (complete drying and fencing for the whole breeding season). While larval removal followed by drying was a successful method of Bd elimination, the effect was only temporary. Since terrestrial habits of adult A. obstetricans prevent them from infection, our findings suggest that, even in simple breeding habitats where all aquatic amphibian stages can be handled and extreme habitat intervention is possible, Bd cannot be eliminated without controlling other potential Bd reservoirs in the surroundings of breeding sites.

A first screening of chytrid fungus Batrachochytrium in amphibians in French zoos

Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) screening was carried out in captive amphibians in order to establish the presence of these fungal pathogens in French zoos. Over a period of 3 years, 437 skin swabs were collected at eight French zoos and one private breeding centre [38 species of Anura, 7 species and 2 subspecies of Urodela (salamanders and newts), and 1 caecilian species] and these were analysed using qPCR. None of the samples revealed the presence of Bsal. Bd was detected four times on three species – American bullfrog Lithobates catesbeianus, Common midwife toad Alytes obstetricans and Yellow‐bellied toad Bombina variegata – all of which originated from or were in contact with the natural environment.

Itraconazole and thiophanate-methyl fail to clear tadpoles naturally infected with the hypervirulent lineage of Batrachochytrium dendrobatidis.

The emerging infectious disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, is a major driver pushing many amphibian species to the brink of extinction. Substantial efforts to develop effective protocols that use antifungal drugs have had notable success. Here, we used the antifungal agents itraconazole and thiophanate-methyl, singly and in combination, in an attempt to treat common midwife toad Alytes obstetricans larvae naturally infected with the globalized hypervirulent lineage of B. dendrobatidis. Despite the successful use of itraconazole in a closely related species (A. muletensis), our results show that these antifungal treatments are not always effective and that full clearance of animals cannot be assumed following treatment.

Infection and transmission heterogeneity of a multi-host pathogen (Batrachochytrium dendrobatidis) within an amphibian community.

The majority of parasites infect multiple hosts. As the outcome of the infection is different in each of them, most studies of wildlife disease focus on the few species that suffer the most severe consequences. However, the role that each host plays in the persistence and transmission of infection can be crucial to understanding the spread of a parasite and the risk it poses to the community. Current theory predicts that certain host species can modulate the infection in other species by amplifying or diluting both infection prevalence and infection intensity, both of which have implications for disease risk within those communities. The fungus Batrachochytrium dendrobatidis (Bd), the causal agent of the disease chytridiomycosis, has caused global amphibian population declines and extinctions. However, not all infected species are affected equally, and thus Bd is a good example of a multi-host pathogen that must ultimately be studied with a community approach. To test whether the common midwife toad Alytes obstetricans is a reservoir and possible amplifier of infection of other species, we used experimental approaches in captive and wild populations to determine the effect of common midwife toad larvae on infection of other amphibian species found in the Peñalara Massif, Spain. We observed that the most widely and heavily infected species, the common midwife toad, may be amplifying the infection loads in other species, all of which have different degrees of susceptibility to Bd infection. Our results have important implications for performing mitigation actions focused on potential 'amplifier' hosts and for better understanding the mechanisms of Bd transmission.

Side effects of itraconazole on post-metamorphic Alytes obstetricans after a cold stress

Itraconazole is the most widely used treatment against Batrachochytrium dendrobatidis (Bd), the fungal pathogen causing chytridiomycosis, a proximate cause of amphibian declines. Several side effects of itraconazole treatment, ranging in severity from depigmentation to death have been reported in different amphibian species and life stages, and these side effects were observed at commonly used dosages of itraconazole. However, no studies have investigated side-effects of itraconazole in conjunction with environmental stress. Post-metamorphic midwife toads (Alytes obstetricans) that were treated with itraconazole and subsequently exposed to a cold stress (exposure to 4°C cold water) had higher mortality rates compared to untreated individuals. Moreover, adults of booroolong frogs (Litoria booroolongensis) treated with itraconazole had a higher probability to become infected when subsequently exposed to Bd. Our results suggest that a post-metamorphosis itraconazole treatment of infected midwife toads combined with a subsequent release into the wild may be an ineffective disease mitigation strategy, as the cold stress during hibernation and/or exposure to Bd in the wild may reduce the hibernation emergence rate of treated individuals in this species.

Laboratory tests of antifungal agents to treat tadpoles against the pathogen Batrachochytrium dendrobatidis

The fungus Batrachochytrium dendrobatidis (Bd), which is the etiological agent of the disease chytridiomycosis, is threatening both wild and captive amphibians. While there are some methods of treating amphibians in captivity, no method has yet been shown to be a promising treatment for amphibian populations in natural habitats. Here we present the results of a laboratory experiment in which we tested 2 antifungal agents that might be used to treat amphibians in the field. As a first step towards the goal of developing mitigation methods, we tested the efficiency of these agents in reducing Bd prevalence and loads (zoospore counts) in the laboratory. We exposed naturally infected tadpoles of the midwife toad Alytes obstetricans to different concentrations of the antifungal agents for 7 d. We found that Virkon Aquatic® affected neither Bd prevalence nor loads. At 0.625 ml l-1 of General Tonic®, prevalence was reduced to 60%, and infected animals had greatly reduced burdens. However, tadpole length was reduced by 19% and mass by 32% on average compared to the control group, suggesting a negative effect on fitness. Tadpole survival was not affected at 0.625 ml l-1 or 1.25 ml l-1, but was reduced to 60% at 2.5 ml l-1. Keeping animals in a dilution of General Tonic® for 7 d at a concentration of 0.625 ml l-1 might be an easy way to reduce zoospore counts in large numbers of animals at relatively low cost.

Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent

Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 μM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 μM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 μM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 μM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 μM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 μg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-β from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.

Populations of a Susceptible Amphibian Species Can Grow despite the Presence of a Pathogenic Chytrid Fungus

Disease can be an important driver of host population dynamics and epizootics can cause severe host population declines. Batrachochytrium dendrobatidis (Bd), the pathogen causing amphibian chytridiomycosis, may occur epizootically or enzootically and can harm amphibian populations in many ways. While effects of Bd epizootics are well documented, the effects of enzootic Bd have rarely been described. We used a state-space model that accounts for observation error to test whether population trends of a species highly susceptible to Bd, the midwife toad Alytes obstetricans, are negatively affected by the enzootic presence of the pathogen. Unexpectedly, Bd had no negative effect on population growth rates from 2002–2008. This suggests that negative effects of disease on individuals do not necessarily translate into negative effects at the population level. Populations of amphibian species that are susceptible to the emerging disease chytridiomycosis can persist despite the enzootic presence of the pathogen under current environmental conditions.

Rural road networks as barriers to gene flow for amphibians: Species-dependent mitigation by traffic calming

Population structuring based on mitochondrial DNA variation along the rural landscape of the Trubia valley in North Iberia revealed significant association between road density and genetic distance between populations of two amphibian species, the midwife toad Alytes obstetricans and the palmate newt Lissotriton helveticus. Traffic calming (concentration of flows on minor rural roads at a few highways to decrease volumes and speeds) near urban settlements mitigates the population fragmentation of L. helveticus but not that of A. obstetricans, indicating that even small roads with low-intensity traffic act as barriers for the latter species. We suggest that the construction of passages for amphibians across rural roads would potentially mitigate population fragmentation of endangered species like the anuran A. obstetricans.

Conformational and membrane interaction studies of the antimicrobial peptide alyteserin-1c and its analogue [E4K]alyteserin-1c

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH 2), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria . The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE- d 3 )-H 2O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu 2 and Val 21. Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys 3 and Val 21. Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala 19 do not interact with the micelles.

Potent and rapid bactericidal action of alyteserin-1c and its [E4K] analog against multidrug-resistant strains of Acinetobacter baumannii

The emergence of multidrug-resistant strains of Acinetobacter baumannii (MDRAB) constitutes a serious threat to public health and necessitates the discovery of new types of antimicrobial agents. Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS·NH 2) is a cationic, α-helical peptide that was first isolated from skin secretions of the midwife toad Alytes obstetricans. Synthetic alyteserin-1c displayed potent activity against clinical isolates of MDRAB (minimum inhibitory concentration, MIC = 5–10 μM; minimum bactericidal concentration, MBC = 5–10 μM) while displaying low hemolytic activity against human erythrocytes (LD 50 = 220 μM). Increasing the cationicity of alyteserin-1c by the substitution Glu 4 → Lys enhanced the potency against MDRAB (MIC = 1.25–5 μM; MBC = 1.25–5 μM) as well as decreasing hemolytic activity (HC 50 > 400 μM). More than 99.9% of the bacteria were killed within 30 min by the [E4K] analog at a concentration of 1 × MBC. Increasing the cationicity of [E4K]alyteserin-1c further by the additional substitutions of Ala 8,Val 14 or Ala 18 by l-Lys did not enhance antimicrobial potency. Derivatives of [E4K]alyteserin-1c containing a palmitate group coupled either to α-amino group at the N-terminus or to ɛ-amino group on the Lys 18 residue of the [E4K,A18K] analog retained antimicrobial activity but showed dramatically increased hemolytic activities (>40- and >13-fold, respectively).

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula

We describe the pathology, isolation and characterisation of a virus responsible for an outbreak of a systemic haemorrhagic disease causing high mortality in tadpoles of the common midwife toad Alytes obstetricans in the 'Picos de Europa' National Park in northern Spain. The virus, provisionally designated as the common midwife toad virus (CMTV), was isolated from homogenates of visceral tissue from diseased toad tadpoles following inoculation on epithelioma papulosum cyprini (EPC) cells. Molecular characterisation of the virus, including sequence analysis of the DNA polymerase and major capsid protein genes, showed that the isolated virus was a ranavirus with marked sequence identity to other members of the genus Ranavirus. A rabbit antiserum raised against purified virions was prepared and used to definitively demonstrate systemic distribution of the virus in diseased tadpoles, indicating that the isolated virus was the primary pathogen.

The alyteserins: Two families of antimicrobial peptides from the skin secretions of the midwife toad Alytes obstetricans (Alytidae)

Two families of structurally related C-terminally α-amidated antimicrobial peptides have been identified in norepinephrine-stimulated skin secretions of the midwife toad Alytes obstetricans (Alytidae). The alyteserin-1 peptides (Gly-Leu-Lys-(Asp/Glu)-Ile-Phe-Lys-Ala-Gly-Leu-Gly-Ser-Leu-Val-Lys-(Gly/Asn)-Ile-Ala-Ala-His-Val-Ala-(Asn/Ser).NH 2) show limited structural similarity to the ascaphins from the skins of frogs of the family Leiopelmatidae. Alyteserin-2a (Ile-Leu-Gly-Lys-Leu-Leu-Ser-Thr-Ala-Ala-Gly-Leu-Leu-Ser-Asn-Leu.NH 2) and alyteserin-2b and -2c (Ile-Leu-Gly-Ala-Ile-Leu-Pro-Leu-Val-Ser-Gly-Leu-Leu-Ser-(Asn/Ser)-Lys-Leu.NH 2) show limited sequence identity with bombinin H6, present in the skins of frogs of the family Bombinatoridae. The alyteserin-1 peptides show selective growth inhibitory activity against the Gram-negative bacteria Escherichia coli (MIC = 25 μM) whereas alyteserin-2a is more potent against the Gram-positive bacteria Staphylococcus aureus (MIC = 50 μM). The hemolytic activity against human erythrocytes of all peptides tested is relatively weak (LC 50 > 100 μM). The data demonstrate that the frogs belonging to the family Alytidae are among those producing dermal antimicrobial peptides that may represent a component of the animal's system of innate immunity.

Influence of advertisement calls on reproductive success in the male midwife toad Alytes obstetricans

AbstractConsidering the advertisement call in anuran as a sexually selected trait, we investigated the effects of male call parameters on reproductive success in midwife toad Alytes obstetricans. The pattern of advertisement call was studied in 81 males. The mean fundamental frequency varied among males. Fundamental frequency, call duration and male size correlated with the male mating success. From the stepwise regression analysis, the low call frequency was found to influence significantly the number of carried eggs and the hatching success. The fact that a single male may fertilise the eggs of several females and a single female may mate with several males constitutes an original sexual system. Large males obtained more matings and showed a higher hatching success. Female choice for dominant frequency may be regarded as a related-fitness trait being correlated with male size. Anyway, the male size and the female choice for low calls result in a same evolutionary trend favouring a best fitness.