Rana Temporaria Research Articles

Electrocardiography effects of Abelmoschus esculentus fruit extract and its isolated compounds using isolated frog heart perfusion

This study evaluated the electrocardiograph effects of Abelmoschus esculentus and its isolated compounds on Rana temporaria (frog) heart. Cardiovascular disease is one of the major causes of mortality which contribute to about 57 million deaths as disclosed by WHO in 2002. There have been many therapeutic approaches to reduce the death rate from cardiovascular disease which include the use of herbal drugs from plants. The whole fruit of Abelmoschus esculentus (Okra) fruit has been used for culinary purposes in Africa. Medicinal properties of Abelmoschus esculentus reported in literature include its use as anti-diabetics, antimicrobial, diuretic agent, and for plasma replacement. However, there have been little or no report on the cardiovascular activities of the extract or isolated compounds from A. esculentus. A. esculentus (Okra) fruits were collected, identified, dried, and powdered. The powdered was extracted using cold maceration with 100% methanol for 72 hours. The crude extract was subjected to repeated fractionation to isolate four (4) compounds. The cardiovascular activities of the crude extract and its isolated compounds were investigated using isolated heart perfusion technique on Rana temporaria (frog) heart. The effects of the extract and isolated compounds on the rate (frequency) and force of contraction (amplitude) of the heart beat were evaluated. The isolated compounds were subjected to spectroscopic analysis using ultraviolent (UV), nuclear magnetic resonance (NMR) (1D and 2D experiments), and mass spectrometry. The compounds were elucidated as quercetin glycoside, quercetin diglycoside, urs-12-ene-3-O-β-D-glucopyranoside and 3-hydroxy-2,3-dihydroimidazo [1,5-a] pyridin-8(5H)-one-5-β-glucopyranoside (esculentoside). The extract and the isolated compounds showed positive inotropic effect on the heart (increase in amplitude of the heart) while they all also possess negative chronotropic effect (decrease in frequency) except compound 4 which possess positive chronotropic effect. This study isolated three different classes of compounds from A. esculentus namely Flavonoids (Quercetin glycoside), Triterpene (Ursolic acid) and pyridine-imidazole (esculentoside). This study also demonstrated that A. esculentus extract and compounds isolated from it has negative chronotropic effects and also possess positive inotropic effects on the heart of R. temporaria.

Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A–L, as an effective chemical dermal defense. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A, but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

Larger is not better: no mate preference by European common frog (Rana temporaria) males

Abstract In explosive breeding frogs, high intrasexual competition between males leads to a sexual coercion ruled mating system, where males presumably evolved preferences for specific female traits. We tested these preferences in the European Common Frog by excluding intrasexual competition. We hypothesized that all males show preferences towards larger female body size, due to higher fecundity. Our results did not show any preference considering female body size, neither in the attempt to amplex a female nor during the formation of pairs. Additionally, we witnessed a high failure rate of male mating attempts, which hints at high mating costs and offers an explanation for the lack of preferences in males. Nonetheless, we observed a non-random mating pattern in successfully formed pairs, where in the absence of size dimorphism females were on average larger than males. This indicates a different mechanism for selection which is independent from male mating preference or scramble competition.

Metamorphic common toads keep chytrid infection under control, but at a cost

AbstractBatrachochytrium dendrobatidis (Bd) causes chytridiomycosis, an infectious disease of amphibians, which has contributed to population declines in hundreds of species worldwide. Common toads (Bufo bufo) exhibit low resistance and relatively high tolerance to Bd infection, which may partly be attributable to bufadienolide toxins secreted in their granular skin glands. Bufadienolides are known to provide an effective defense against several pathogens, parasites, and predators. The toxin production of bufonids is a plastic trait, inducible by several environmental factors. Here, we experimentally infected juvenile common toads with Bd and investigated if the toadlets could clear the infection over time, whether the infection induced bufadienolide production, and whether the infection caused decreased body mass. We found that prevalence remained 100% throughout the entire experimental period, but infection intensity did not increase and it was significantly lower on day 30 than on day 20. At the same time, compared to controls, infected toadlets produced lesser amounts of bufadienolides and their body mass was also lower. These results suggest that although young toadlets may not be able to clear Bd infection on their own, they may be able to keep infection intensities under control. Nonetheless, even if toadlets do not succumb to the disease, the costs of chronic infection may still compromise their fitness.

The Arsenic-Antimony Creek at Sauerbrunn/Burgenland, Austria: A Toxic Habitat for Amphibians.

(1) Background: All Austrian amphibians are affected by the degradation of habitats. Mining contributes to habitat destruction by the formation of spoil heaps and mine drainage waters. In Stadtschlaining/Burgenland, antimony mining led to increased arsenic (As) and antimony (Sb) concentrations in soil and water. This study investigates a contaminated creek, still inhabited by amphibians. (2) Methods: Water and soil were analyzed along the creek and correlated with the occurrence of amphibians. (3) Results: As and Sb were increased, with up to 49,000 mg/kg As and 2446 mg/kg Sb in the soil. Up to 317 mg/kg As and 156 mg/kg Sb became bioavailable under gastric, and up to 298 mg/kg As and 30 mg/kg Sb under intestinal conditions, and were absorbed upon ingestion of soil. Larvae of Salamandra salamandra were found throughout the creek; survival rates were low. Rana temporaria occurs in the most contaminated sections but does not propagate here. Bombina variegata appears occasionally. Amphibians seem not to be able to detect and avoid metal or metalloid contamination. (4) Conclusion: Survival of larvae is dubious, but adult amphibians survive without apparent damage under severe metalloid contamination.

Different time patterns of the presence of red-eared slider influence the ontogeny dynamics of common frog tadpoles

The coexistence of species in a given community depends on the set of species involved and the timing of their interactions. Many native communities are increasingly forced to face both direct and indirect pressures from new alien predators, which, in extreme cases, can lead to the extinction of prey populations. In this study, we examine the dynamics of the ontogeny of common frog (Rana temporaria) tadpoles under different time patterns of an alien predator—the red-eared slider (Trachemys scripta elegans) presence. We found that the tadpoles had a longer larval period and were smaller in size at metamorphosis and lower in body mass when the predator was present in early development than when the tadpoles developed without a predator. The early presence of a predator conspicuously reduced the growth increments of the tadpoles at early development. After the removal of the predator, growth accelerated above the level measured under the conditions of both the late predator and no predator. However, these growth rates did not exceed the growth rates of equally sized tadpoles in the other treatments and therefore were not sufficient to compensate for the growth slowdown in the first part of development. The presence of a predator in late tadpole development influenced neither the time to metamorphosis nor size/body mass at metamorphosis. In conclusion, the predator had the effect on metamorphosis traits only if it was present in the early development of tadpoles.

“Heat waves” experienced during larval life have species-specific consequences on life-history traits and sexual development in anuran amphibians

Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimize risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured 2 months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated.

Long-Term Changes in Four Populations of the Spiny Toad, Bufo spinosus, in Western France; Data from Road Mortalities

Habitat fragmentation is widely recognized as a contributor to the decline of biodiversity, with amphibians one of the key groups impacted. To understand the effects of habitat fragmentation on amphibian populations requires long-term data sets showing population trends. In this paper, road mortalities were employed as proxies to describe long-term numbers of four populations of the spiny toad Bufo spinosus in western France during a 17-year period. Road mortalities were found during all months in all populations but were most frequent during October, November and December, the main migratory period. Large females were found significantly more frequently during these migration months, forming 45% of the total sample, compared with their presence from January to September (34.4%). The long-term trends were evaluated using regression analysis of the logarithmic (loge) transforms of annual counts as dependent variables against year as the independent variables. All coefficients showed no significant departure from the 0 hypothetical coefficients, indicative of population stability. This was supported by jackknife analysis, which showed good agreement of the pseudo-regression coefficients with the true equations. Stepwise regression of potential climate impacts on toad numbers suggested rainfall levels in October adjusted to 2- and 3-year lags were involved in driving population change. Road mortality counts were also made during 2020 and 2021 when human movement restrictions were in place due to the COVID-19 pandemic. To estimate the potential impact on this disturbance in the methodology, the Poisson distribution was used to estimate potential differences between what would have been expected counts and the observed counts. The results indicate that the observed mortalities were significantly lower than expected in all four populations.

How does the feeding behaviour of the common forest toad Rhinella henseli (Anura: Bufonidae) vary in space and time? Trophic ecology, chemical and antimicrobial activity

Studies in trophic and chemical ecology, in particular in amphibians, have gained increasing attention in recent years, given that this is the vertebrate group that has suffered the greatest decline in recent years, caused by the degradation of natural ecosystems and emerging diseases. The assessment of food preferences and prey availability between areas and seasons provides important parameters for the understanding of the population dynamics of leaf-litter toads. The study of the secretions of the parotoid macroglands of these toads also provides insights into the role of these secretions in fighting frog pathogens and their potential applications to combat pathogens that are harmful to humans. In the present study, we describe the trophic ecology of Rhinella henseli (Lutz, 1934), and the variation in its diet between seasons and areas. We also attempt to identify the chemical composition of the secretions of the parotoid macrogland found in the parotoid glands and test their potential antimicrobial activity. We sampled two toad populations in the Atlantic Forest of southern Brazil. The composition of the diet was analysed by season (warm vs. cool) and study area, with the prey items being identified to genus, whenever possible, and classified using the Index of Relative Importance. The parotoid secretions were removed manually from the parotoid glands and analysed via HPLC-MS/MS. We ran microdilution and agar plug diffusion tests to assess antimicrobial activity. The principal prey of these toads are large ants, primarily Pachycondyla sp., which vary in abundance between seasons and, to a lesser extent, between areas. We identified 21 chemical compounds, primarily steroidal bufadienolides. One of the populations presented a subset of 14 of these 21 compounds, reflecting the variation in their spatial distribution. These compounds presented anti-pathogenic properties against Candida albicans and, to a lesser extent, Staphylococcus saprophyticus. Our results indicate that the diet of R. henseli varies significantly between areas and seasons, as do the secretions of their parotoid macroglands between areas. The toxins exhibit antimicrobial activity, although the compounds must be tested in isolation to confirm this.

EDNA Metabarcoding Benchmarked towards Conventional Survey Methods in Amphibian Monitoring.

Simple SummaryAmphibian species are declining worldwide, and precise monitoring is key to ensuring timely protection and thereby ceasing deteriorating populations. Conventional monitoring methods are invasive, time-consuming, and dependent on expert knowledge. eDNA methods have been suggested as a replacement for or supplement to conventional survey methods. The present study assessed amphibian detection of conventional survey methods and eDNA metabarcoding in Danish lakes and ponds to address how the application of eDNA surveys can supplement the currently applied methodology. The study found eDNA metabarcoding to detect five out of six species detected through conventional methods. Furthermore, it is expected that the results in the present study reflect the time of sampling for the applied methods. The findings in the present study indicate that eDNA metabarcoding detects multiple Danish amphibian species and can produce knowledge on the occurrence and distribution for amphibian species. Implementing it as a supplement for conventional survey methods in nature monitoring will enable a higher frequency of monitoring and yield knowledge of species composition.A keystone in protection work is accurate and thorough the monitoring of amphibian species, and the currently applied conventional survey methods are invasive, time-consuming, and dependent on expert knowledge. Research suggests that eDNA metabarcoding is a precise and cost-efficient method that could supplement the currently applied methods. The present study assessed the efficiency of conventional survey methods and eDNA metabarcoding in terms of species richness, the average number of detected species per site, the relative frequency of species occurrence, and the similarity of applied methods. The study found eDNA metabarcoding surveys to detect Lissotriton vulgaris (smooth newt), Triturus cristatus (great crested newt), Rana arvalis (moor frog), Rana temporaria (common frog), and Bufo bufo (common toad), as well as an average of 0.9 species per site, reflecting the species composition at the time of sampling in mid-July 2020. In addition to the species mentioned above, the conventional survey detected Epidalea calamita (natterjack toad) and an average of 1.7 species per site, reflecting the species composition at the time of sampling in early June 2020. The similarity between the methods applied in the present study was 27%, thus indicating a large number of unique observations of both eDNA metabarcoding and conventional surveys. The differences in detection can most likely be explained by the time of sampling, which was conducted a month apart. eDNA metabarcoding was efficient in detecting multiple amphibian species and produced unique observations that were not detected using conventional survey methods. Applying eDNA techniques as a supplement will most likely produce important knowledge on species distribution and presence, as well as enable more frequent monitoring due to cost efficiency and disturbance.

Characterization of Some Novel Antimicrobial Peptides from African Common Toad, Sclerophrys regularis

The growing number of antimicrobial resistant bacteria have necessitated urgent need for newer and effective antimicrobials. Antimicrobial peptides (AMPs) which are also called host defense peptides are promising class of bioactives found in all living thing especially amphibians. A total of 355 Sclerophrys regularis were collected and each toad was weighed and 0.02mL/g of epinephrine (1mg/mL) was administered via the dorsal lymph sac of the toad. The skin secretions were washed, lyophilized and stored at -20°C. The secretion was purified using Sephadex G-50 gel purification column. The fractions showing antimicrobial activities against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella typhii ATCC 14028, Citrobacter freundii ATCC 8090, Staphylococcus aureus ATCC 25913 and Candida albicans ATCC 3147 were further purified by RP- HPFC. The purified fractions were lyophilized and m/z ratio peaks were determined using MALDI TOF/TOF MS. The sequences corresponding to the peaks were determined using Mascot Peptide Mass Fingerprint which revealed novel AMP sequences: IHAGKTVPIVK, MHLLWR and LTGQIKNGLSGR which were named Regularin-2, Regularin-3 and Regularin-4 respectively. The MICs of purified fractions were between 50 – 200 µg/mL and the antimicrobial activities were maintained when stored at -20 °C for 180 days and was significant at p < 0.05. Bioprospecting of Nigerian toad specie (S. regularis) could yield potential therapeutic AMP agents.

Effects of sodium perchlorate and 6-propylthiouracil on metamorphosis and thyroid gland histopathology in the European common frog (Rana temporaria).

Several chemicals have been identified as thyroid hormone axis disrupting chemicals (THADCs) able to interfere with the thyroid hormone system during fetal life and early life stages, thereby impairing neurodevelopment in mammals and inducing development and growth disorders in fish and amphibians. However, identification of THADCs is particularly challenging, and thyroid modalities are currently only assessed in vivo by mammalian and amphibian tests. The aquatic African clawed frog (Xenopus laevis/tropicalis) is the model species of the amphibian test guidelines developed by the OECD and the United States Environmental Protection Agency, but as most European amphibians are semi-aquatic, concern has been raised whether the sensitivity of native European species is comparable to Xenopus. A shortened version of the OEDC test guideline 241 (Larval Amphibian Growth and Development Assay, LAGDA) was used to investigate the effects of two model THADCs on the metamorphosis and thyroid histopathology in the European common frog (Rana temporaria). R. temporaria eggs were collected on the field and exposed till metamorphic climax to sodium perchlorate (11.9-426.5 μg/L perchlorate concentrations) and 6-propylthiouracil (PTU: 1.23-47.7mg/L). PTU severely delayed metamorphosis and affected several thyroid gland histopathological endpoints at slightly lower concentrations compared to Xenopus. As opposed to what was described in similar Xenopus studies, we observed no effect of perchlorate on the investigated endpoints. Interspecies differences may be linked to mechanisms of action.

Thermal tolerance and acclimation capacity in the European common frog (Rana temporaria) change throughout ontogeny.

Phenotypic plasticity may allow ectotherms with complex life histories such as amphibians to cope with climate-driven changes in their environment. Plasticity in thermal tolerance (i.e., shifts of thermal limits via acclimation to higher temperatures) has been proposed as a mechanism to cope with warming and extreme thermal events. However, thermal tolerance and, hence, acclimation capacity, is known to vary with life stage. Using the common frog (Rana temporaria) as a model species, we measured the capacity to adjust lower (CTmin ) and upper (CTmax ) critical thermal limits at different acclimation temperatures. We calculated the acclimation response ratio as a metric to assess the stage-specific acclimation capacity at each of seven consecutive ontogenetic stages and tested whether acclimation capacity was influenced by body mass and/or age. We further examined how acclimation temperature, body mass, age, and ontogenetic stage influenced CTmin and CTmax . In the temperate population of R. temporaria that we studied, thermal tolerance and acclimation capacity were affected by the ontogenetic stage. However, acclimation capacity at both thermal limits was well below 100% at all life stages tested. The lowest and highest acclimation capacity in thermal limits was observed in young and late larvae, respectively. The relatively low acclimation capacity of young larvae highlights a clear risk of amphibian populations to ongoing climate change. Ignoring stage-specific differences in thermal physiology may drastically underestimate the climate vulnerability of species, which will hamper successful conservation actions.

Challenging a host-pathogen paradigm: Susceptibility to chytridiomycosis is decoupled from genetic erosion.

The putatively positive association between host genetic diversity and the ability to defend against pathogens has long attracted the attention of evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades as a cause of dramatic declines and extinctions across the amphibian clade. Bd susceptibility can vary widely across populations of the same species, but the relationship between standing genetic diversity and susceptibility has remained notably underexplored so far. Here, we focus on a putatively Bd‐naive system of two mainland and two island populations of the common toad (Bufo bufo) at the edge of the species’ range and use controlled infection experiments and dd‐RAD sequencing of >10 000 SNPs across 95 individuals to characterize the role of host population identity, genetic variation and individual body mass in mediating host response to the pathogen. We found strong genetic differentiation between populations and marked variation in their susceptibility to Bd. This variation was not, however, governed by isolation‐mediated genetic erosion, and individual heterozygosity was even found to be negatively correlated with survival. Individual survival during infection experiments was strongly positively related to body mass, which itself was unrelated to population of origin or heterozygosity. Our findings underscore the general importance of context‐dependency when assessing the role of host genetic variation for the ability of defence against pathogens.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex‐ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex‐reversing stimuli more frequently, which may lead to higher sex‐reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR‐based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex‐reversal frequency in free‐living adults despite the presence of various endocrine‐disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L−1 17α‐ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L−1 EE2 and a glyphosate‐based herbicide with 3 µg L−1 or 3 mg L−1 glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex‐reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species‐specific resistance to sex reversal.

First record of ranavirus (Ranavirus sp.) in Siberia, Russia

Ranaviruses are a group of double-strand DNA viruses that infect fish, amphibians and reptiles. These viruses are responsible for mass fish and amphibian mortality events worldwide, both in the wild and at fish and amphibian farms. The number of detected epizootics has grown significantly in recent years. In Eastern Europe and Northern Asia, including Russia, very few ranavirus monitoring studies have been conducted, in contrast with Western Europe and America. In the present work, we used a qPCR assay to survey for the first time the amphibian populations of West Siberia (Russia) for the presence of ranaviruses. In total, we studied 252 tissue samples from six amphibian species, collected across West Siberia from the south to the Arctic regions. We report a single infected sample: a common toad (Bufo bufo) captured near Tyumen city. The phylogenetic analysis showed that the detected virus strain belongs to the CMTV lineage. This is only the second observation of Ranavirus in Russia.

Temperature and duration of exposure drive infection intensity with the amphibian pathogen Batrachochytrium dendrobatidis.

The intensity of a pathogen infection plays a key role in determining how the host responds to infection. Hosts with high infections are more likely to transmit infection to others, and are may be more likely to experience progression from infection to disease symptoms, to being physiologically compromised by disease. Understanding how and why hosts exhibit variation in infection intensity therefore plays a major part in developing and implementing measures aimed at controlling infection spread, its effects, and its chance of persisting and circulating within a population of hosts. To track the relative importance of a number of variables in determining the level of infection intensity, we ran field-surveys at two breeding sites over a 12 month period using marked larvae of the common midwife toad (Alyes obstetricans) and their levels of infection with the amphibian pathogen Batrachochytrium dendrobatidis (Bd). At each sampling occasion we measured the density of larvae, the temperature of the water in the 48 h prior to sampling, the period of time the sampled individual had been in the water body, the developmental (Gosner) stage and the intensity of Bd infection of the individual. Overall our data suggest that the temperature and the duration of time spent in the water play a major role in determining the intensity of Bd infection within an individual host. However, although the duration of time spent in the water was clearly associated with infection intensity, the relationship was negative: larvae that had spent less than 3–6 months in the water had significantly higher infection intensities than those that had spent over 12 months, although this infection intensity peaked between 9 and 12 months. This could be due to animals with heavier infections developing more quickly, suffering increased mortality or, more likely, losing their mouthparts (the only part of anuran larvae that can be infected with Bd). Overall, our results identify drivers of infection intensity, and potentially transmissibility and spread, and we attribute these differences to both host and pathogen biology.

Black-spotted pond frog Pelophylax nigromaculatus as a new host for the renal coccidian genus Hyaloklossia (Alveolata: Apicomplexa).

Hyaloklossia Labbé, 1896 (Apicomplexa: Sarcocystidae) is a renal coccidium that infects anuran species. The genus consists of two species: H. lieberkuehni, recorded from Pelophylax kl. esculentus, Pelophylax ridibundus, and Rana temporaria in Europe; and H. kasumiensis, recorded from Pelophylax porosus porosus in Japan. However, there have been no reports of Hyaloklossia in the other anurans in Japan. On June 2021, we examined a total of 58 adult frogs comprising 2 P. p. porosus, 23 Pelophylax nigromaculatus, 8 Rana japonica, 3 Glandirana rugosa (Ranidae), 13 Fejervarya kawamurai (Dicroglossidae), and 9 Buergeria buergeri (Rhacophoridae) for infection by Hyaloklossia. Microscopic examination of kidney tissues revealed a high infection incidence of 47.8% (11/23) in P. nigromaculatus, but the other frog species were negative for Hyaloklossia. Morphological and molecular analyses using nuclear ribosomal and mitochondrial genes confirmed the infective species as H. kasumiensis. This is a new host record for H. kasumiensis.

Nicosulfuron, a sulfonylurea herbicide, alters embryonic development and oxidative status of hatchlings at environmental concentrations in an amphibian species

The widespread use of agrochemicals for controlling pests and diseases of crops is recognized as a main threat to biodiversity. Sulfonylurea herbicides are being increasingly used and display low levels of degradation in water which suggest that they might affect non-target organisms. In a common garden experiment, eggs of a widespread amphibian (Bufo spinosus) were exposed to sublethal environmentally relevant concentrations of a widely used sulfonylurea herbicide, nicosulfuron, during the whole embryonic development. We assessed development-related traits (i.e., development duration, hatching success, hatchling size and occurrence of malformation) as well as antioxidant markers in response to contamination (i.e., SOD, GPx, catalase, thiols and relevant ratios thereof). We found that sublethal concentrations of nicosulfuron increased embryonic development duration, increased hatchling size and tended to increase malformations. Embryos exposed to nicosulfuron displayed decreased thiols and increased catalase activity suggesting alteration of oxidative status. We did not find any effect of nicosulfuron on SOD and GPx levels. Interestingly, higher catalase activity was linked to higher proportion of malformed individuals, suggesting that exposure to nicosulfuron induced teratogenic effects. Our results suggest that alteration of antioxidant levels might be one physiological mechanism through which nicosulfuron might cause detrimental effects on amphibian embryos. Sublethal effects of pesticides at environmentally relevant concentrations have been overlooked and require further investigations, especially in non-target taxa occurring in agricultural landscapes.

Toad invasion of Malagasy forests triggers severe mortality of a predatory snake

The Asian common toad Duttaphrynus melanostictus has been introduced to the eastern province of Toamasina in Madagascar, where it is feared to be having devastating effects on native communities by poisoning frog-eating predators. So far it is unclear whether the toad can invade forest habitats, and empirical evidence of its impact on native predators is lacking. We used radio tracking to investigate the spatial behaviour of adult toads in a small parcel of lowland humid forest and we quantify the disruptive effects of toad poisoning on a native frog-eating snake, the Malagasy cat-eyed snake (Madagascarophis colubrinus). We used N-mixture models to estimate the population size of cat-eyed snakes, and used the mortality events recorded in the same area to obtain an estimate of monthly mortality rate of snakes due to toad poisoning. Our results point to a drastic mortality rate of snakes that, if constant through the year, could halve the predator population, with the potential risk of local extirpation. We expect cat-eyed snake populations across rural and suburban areas of Toamasina to be severely affected by the toad invasion, and suggest that future research should investigate the effects of indirect facilitation of human-commensal rodents and the potential repercussions on human health and well-being.