Dendrobates Tinctorius Research Articles

Early development of the glucocorticoid stress response in poison frog tadpoles.

The timing of HPA axis development differs across species. We studied the HPA axis across tadpole development in Dendrobates tinctorius . No difference in corticosterone concentration across development.No difference in corticosterone response to ACTH across development.Results suggest an early developed HPA axis is essential for their life history.

Individual behavioral variability across time and contexts in Dendrobates tinctorius poison frogs

Individual behavioral variability across time and contexts in Dendrobates tinctorius poison frogs

Genome Assembly of the Dyeing Poison Frog Provides Insights into the Dynamics of Transposable Element and Genome-Size Evolution.

Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius, while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes.

Tap dancing frogs: Posterior toe tapping and feeding in Dendrobates tinctorius

AbstractAnimals have myriad adaptations to help them hunt and feed in the most efficient and effective manner. One mysterious behavior related to hunting and feeding is the posterior toe tapping behavior of some frogs. Biologists and hobbyists alike have long noticed this behavior, but there is little empirical data to explain its causes and consequences. To test the hypothesis that tapping is related to feeding and modulated by environmental context, we conducted a series of related experiments in the Dyeing poison frog, Dendrobates tinctorius. We first confirmed that tap rate was higher during feeding as has been observed in other species. Interestingly, this effect was heightened in the presence of a conspecific. We next asked whether frogs tapped less under conditions when prey were visible, but inaccessible. Finally, we asked whether D. tinctorius adjusted tap rate based on substrate characteristics and whether prey capture success was higher when tapping. In addition to confirming an association between tapping and feeding, our work demonstrates modulation of toe tapping based on social context, prey accessibility, and substrate characteristics. Based on our findings, we suggest that tapping could act to induce prey movement and thereby facilitate prey detection and capture by frogs.

Body condition and diet of the dyeing poison frog Dendrobates tinctorius (Cuvier 1797) (Amphibia: Dendrobatidae) from the eastern Brazilian Amazonia

The present study adds new data on the natural history of Dendrobatestinctorius. We conducted our fieldwork during the rainy season of the years2018 and 2019 in two protected areas (Parque Natural Municipal do Cancão–PNMC and Reserva Extrativista Beija-Flor Brilho de Fogo–REBFBF) in the state of Amapá, Brazil. Through active visual searches we sampled 22 individuals of D. tinctorius, of which 10 were countedfor the population of PNMC and 12 for the population of REBFBF. We calculatedthe Body Condition Index (BCI) for all individuals and, for dietary data, each frog’s stomach was flushed. Females from the REBFBF were larger than males whencompared to the PNMC population. BCI of females from the REBFBF showed higher values than males. Gravid females of both populations showed higher BCI. Retrievedstomachs revealed a total of 290 prey items divided into four main taxonomiccategories of Arthropods. The Index of Relative Importance revealed that antsof the genus Solenopsis were the majority of the diet, and mites represented only 4.86%. The niche breadth for both populations was narrow, indicating a specialist diet, as expected for the species. Our study evidences the importance of integration between BCI and dietary aspects.

Intraspecific divergence of sexual size dimorphism and reproductive strategies in a polytypic poison frog

AbstractIntraspecific variation in body size, both among populations and between sexes, is an important factor influencing life-history strategies. This variation might be the response to different environmental conditions, as well as natural and sexual selection, and can result in differences in behavior and reproductive strategies among populations. Here, we use the dyeing poison frog (Dendrobates tinctorius) as a model to investigate how interpopulation variation in body size and sexual size dimorphism affects reproductive strategies. As body size increased, sexual size dimorphism also increased, i.e., females were larger than males, and more so in populations with overall larger frogs. This indicates that there is a stronger selection for body size in females than in males, likely as a response to divergent reproductive investment between the sexes. Females from larger-bodied populations produced larger clutches, but the overall number of froglets produced per clutch did not differ among populations. We discuss potential causes and mechanisms that might be responsible for the observed divergence in body size, sexual size dimorphism, and reproductive strategies among populations that likely represent local adaptations. Our findings demonstrate the importance of cross-population studies, cautioning against drawing general conclusions about a species’ ecology without accounting for intraspecific variation.

Visual environment of rearing sites affects larval response to perceived risk in poison frogs.

Turbidity challenges the visual performance of aquatic animals. Here, we use the natural diversity of ephemeral rearing sites occupied by tadpoles of two poison frog species to explore the relationship between environments with limited visibility and individual response to perceived risk. To compare how species with diverse natural histories respond to risk after developing in a range of photic environments, we sampled wild tadpoles of (1) Dendrobates tinctorius, a rearing-site generalist with facultatively cannibalistic tadpoles and (2) Oophaga pumilio, a small-pool specialist dependent on maternal food-provisioning. Using experimental arenas, we measured tadpole activity and space use first on a black and white background, and then on either black or white backgrounds where tadpoles were exposed to potentially predatory visual stimuli. The effects of rearing environment on D. tinctorius tadpoles were clear: tadpoles from darker pools were less active than tadpoles from brighter pools and did not respond to the visual stimuli, whereas tadpoles from brighter pools swam more when paired with conspecifics versus predatory insect larvae, suggesting that tadpoles can visually discriminate between predators. For O. pumilio, tadpoles were more active on experimental backgrounds that more closely matched the luminosity of their rearing sites, but their responses to the two visual stimuli did not differ. Larval specialisation associated with species-specific microhabitats may underlie the observed responses to visual stimuli. Our findings demonstrate that light availability in wild larval rearing conditions influences risk perception in novel contexts, and provides insight into how visually guided animals may respond to sudden environmental disturbances.

Linking Predator Responses to Alkaloid Variability in Poison Frogs.

Many chemically-defended/aposematic species rely on diet for sequestering the toxins with which they defend themselves. This dietary acquisition can lead to variable chemical defenses across space, as the community composition of chemical sources is likely to vary across the range of (an aposematic) species. We characterized the alkaloid content of two populations of the Dyeing Poison Frog (Dendrobates tinctorius) in northeastern French Guiana. Additionally, we conducted unpalatability experiments with naive predators, Blue Tits (Cyanistes caeruleus), using whole-skin secretion cocktails to assess how a model predator would respond to the defense of individuals from each population. While there was some overlap between the two D. tinctorius populations in terms of alkaloid content, our analysis revealed that these two populations are markedly distinct in terms of overall alkaloid profiles. Predator responses to skin secretions differed between the populations. We identified 15 candidate alkaloids (including three previously undescribed) in seven classes that are correlated with predator response in one frog population. We describe alkaloid profile differences between populations for D. tinctorius and provide a novel method for assessing unpalatability of skin secretions and identifying which toxins may contribute to the predator response. In one population, our results suggest 15 alkaloids that are implicated in predator aversive response. This method is the first step in identifying the causal link between alkaloids and behavioral responses of predators, and thus makes sense of how varying alkaloid combinations are capable of eliciting consistent behavioral responses, and eventually driving evolutionary change in aposematic characters (or characteristics).

Poison frog dietary preference depends on prey type and alkaloid load.

The ability to acquire chemical defenses through the diet has evolved across several major taxa. Chemically defended organisms may need to balance chemical defense acquisition and nutritional quality of prey items. However, these dietary preferences and potential trade-offs are rarely considered in the framework of diet-derived defenses. Poison frogs (Family Dendrobatidae) acquire defensive alkaloids from their arthropod diet of ants and mites, although their dietary preferences have never been investigated. We conducted prey preference assays with the Dyeing Poison frog (Dendrobates tinctorius) to test the hypothesis that alkaloid load and prey traits influence frog dietary preferences. We tested size preferences (big versus small) within each of four prey groups (ants, beetles, flies, and fly larvae) and found that frogs preferred interacting with smaller prey items of the fly and beetle groups. Frog taxonomic prey preferences were also tested as we experimentally increased their chemical defense load by feeding frogs decahydroquinoline, an alkaloid compound similar to those naturally found in their diet. Contrary to our expectations, overall preferences did not change during alkaloid consumption, as frogs across groups preferred fly larvae over other prey. Finally, we assessed the protein and lipid content of prey items and found that small ants have the highest lipid content while large fly larvae have the highest protein content. Our results suggest that consideration of toxicity and prey nutritional value are important factors in understanding the evolution of acquired chemical defenses and niche partitioning.

Noninvasive Detection of Chemical Defenses in Poison Frogs Using the MasSpec Pen.

Poison frogs are well-known for their fascinating ability to store alkaloids in their skin as chemical defense against predators. Chemical methods used to study these alkaloids are limited by requirements for euthanasia or stress during sampling. Here, we demonstrate sensitive and biocompatible alkaloid detection and monitoring in vivo using the MasSpec Pen, a handheld, noninvasive chemical detection device coupled to a mass spectrometer. The MasSpec Pen allowed rapid (<15 s), gentle, and consecutive molecular analysis without harm or undue stress to the animals. Through a month-long alkaloid-feeding study with the dyeing poison frog, we observed temporal dynamics of chemical sequestration in vivo by comparing frogs fed the alkaloid decahydroquinoline (DHQ) to vehicle-fed frogs. We also demonstrate the feasibility of the MasSpec Pen for the untargeted detection of rich alkaloid profiles from skin extracts of the Diablito poison frog, collected from two distinct geographical populations in Ecuador. The results obtained in this study demonstrate the utility of the MasSpec Pen for direct, rapid, and biocompatible analysis of poison frog alkaloids.

Size-dependent aggression towards kin in a cannibalistic species.

In juveniles extreme intraspecies aggression can seem counter-intuitive, as it might endanger their developmental goal of surviving until reproductive stage. Ultimately, aggression can be vital for survival, although the factors (e.g., genetic or environmental) leading to the expression and intensity of this behavior vary across taxa. Attacking (and sometimes killing) related individuals may reduce inclusive fitness; as a solution to this problem, some species exhibit kin discrimination and preferentially attack unrelated individuals. Here, we used both experimental and modeling approaches to consider how physical traits (e.g., size in relation to opponent) and genetic relatedness mediate aggression in dyads of cannibalistic Dendrobates tinctorius tadpoles. We paired full-sibling, half-sibling, and non-sibling tadpoles of different sizes together in an arena and recorded their aggression and activity. We found that the interaction between relative size and relatedness predicts aggressive behavior: large individuals in non-sibling dyads are significantly more aggressive than large individuals in sibling dyads. Unexpectedly, although siblings tended to attack less overall, in size-mismatched pairs they attacked faster than in non-sibling treatments. Using a theoretical model to complement these empirical findings, we propose that larval aggression reflects a balance between relatedness and size where individuals trade-off their own fitness with that of their relatives.

Molecular physiology of pumiliotoxin sequestration in a poison frog.

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Naive poison frog tadpoles use bi-modal cues to avoid insect predators but not heterospecific predatory tadpoles.

ABSTRACTFor animals to survive until reproduction, it is crucial that juveniles successfully detect potential predators and respond with appropriate behavior. The recognition of cues originating from predators can be innate or learned. Cues of various modalities might be used alone or in multi-modal combinations to detect and distinguish predators but studies investigating multi-modal integration in predator avoidance are scarce. Here, we used wild, naive tadpoles of the Neotropical poison frog Allobates femoralis ( Boulenger, 1884) to test their reaction to cues with two modalities from two different sympatrically occurring potential predators: heterospecific predatory Dendrobates tinctorius tadpoles and dragonfly larvae. We presented A. femoralis tadpoles with olfactory or visual cues, or a combination of the two, and compared their reaction to a water control in a between-individual design. In our trials, A. femoralis tadpoles reacted to multi-modal stimuli (a combination of visual and chemical information) originating from dragonfly larvae with avoidance but showed no reaction to uni-modal cues or cues from heterospecific tadpoles. In addition, visual cues from conspecifics increased swimming activity while cues from predators had no effect on tadpole activity. Our results show that A. femoralis tadpoles can innately recognize some predators and probably need both visual and chemical information to effectively avoid them. This is the first study looking at anti-predator behavior in poison frog tadpoles. We discuss how parental care might influence the expression of predator avoidance responses in tadpoles.

Pool choice in a vertical landscape: Tadpole-rearing site flexibility in phytotelm-breeding frogs.

Many species of Neotropical frogs have evolved to deposit their tadpoles in small water bodies inside plant structures called phytotelmata. These pools are small enough to exclude large predators but have limited nutrients and high desiccation risk. Here, we explore phytotelm use by three common Neotropical species: Osteocephalus oophagus, an arboreal frog that periodically feeds eggs to its tadpoles; Dendrobates tinctorius, a tadpole‐transporting poison frog with cannibalistic tadpoles; and Allobates femoralis, a terrestrial tadpole‐transporting poison frog with omnivorous tadpoles. We found that D. tinctorius occupies pools across the chemical and vertical gradient, whereas A. femoralis and O. oophagus appear to have narrower deposition options that are restricted primarily by pool height, water capacity, alkalinity, and salinity. Dendrobates tinctorius tadpoles are particularly flexible and can survive in a wide range of chemical, physical, and biological conditions, whereas O. oophagus seems to prefer small, clear pools and A. femoralis occupies medium‐sized pools with abundant leaf litter and low salinity. Together, these results show the possible niche partitioning of phytotelmata among frogs and provide insight into stressors and resilience of phytotelm breeders.

Effect of Manual Restraint and Visual Security on Heart Rate in Dyeing Poison Dart Frogs (Dendrobates tinctorius azureus) and Leopard Frogs (Lithobates pipiens)

Frogs are popular exotic pets and research subjects. Manual restraint is routinely used for basic procedures in frogs, including examination and sample collection, but there is little information on its physiologic effects in amphibians. Previous literature suggested that amphibians lack emotional responses and, therefore, consciousness, based upon an inability to elicit stress tachycardia with gentle handling. To determine if manual restraint results in a stress tachycardia in frogs, 10 poison dart frogs (Dendrobates tinctorius) and 8 leopard frogs (Lithobates pipiens) were manually restrained and heart rate (HR) recorded at both the pectoral girdle and pelvic patch using a Doppler flow probe. Frogs were randomly placed in one of two (opaque versus translucent) types of plastic containers for 22 min and HR measured every 2 min through the bottom of the container using the frogs' pelvic patch area. Frogs were then removed from the container, manually restrained again, and HR recorded. Heart rate decreased significantly after the frogs were placed in the containers following manual restraint, and increased significantly for the second restraint period. There were no significant differences in measured HR between anatomic sites (pelvic vs. pectoral) or container types. Contrary to previous descriptions, these results demonstrated that, similar to other mammal and reptile species, frogs demonstrated stress tachycardia during manual restraint, and, therefore, clinicians and researchers should be mindful of induced stress when restraining amphibians.

Evaluation of Subcutaneous Alfaxalone for Sedation in Juvenile Blue Poison Dart Frogs (Dendrobates tinctorius azureus)

Blue poison dart frogs (Dendrobates tinctorius azureus) are commonly found in zoological institutions and are becoming popular in the pet trade. Sedation or light anesthesia is required for safe and effective handling of this species. In this study, the effect of subcutaneous administration of alfaxalone at two different doses (20 and 30 mg/kg) was evaluated in juvenile blue poison dart frogs (N = 10) in a prospective, randomized, blinded, crossover study. Heart rate, pulmonic respiratory rate, various reflexes, and behavioral parameters were monitored after drug administration. Both protocols resulted in rapid induction of sedation (median [range]) with loss of righting reflex: 20 mg/kg, 10 (10–20 min) and 30 mg/ kg, 10 (10–10 min). The time (mean ± SD) to return to spontaneous movement (20 mg/kg, 39.6 ± 7.6 min and 30 mg/kg, 54.5 ± 9.9 min; P < 0.01) and full recovery (20 mg/kg, 42.6 ± 8 min and 30 mg/kg, 61.6 ± 9.1 min; P < 0.01) after alfaxalone administration were significantly longer in the 30-mg/kg group. The frogs in both groups continued to remain responsive to tactile stimulation and retained palpebral reflexes. Alfaxalone induced cardiopulmonary depression and periods of apnea at both doses. The subcutaneous administration of 20 and 30 mg/kg alfaxalone resulted in clinically effective sedation in blue poison dart frogs. Sedation levels achieved in this prospective study would be adequate for a variety of noninvasive clinical procedures.

Visible implant elastomer (VIE) success in early larval stages of a tropical amphibian species.

Animals are often difficult to distinguish at an individual level, and being able to identify individuals can be crucial in ecological or behavioral studies. In response to this challenge, biologists have developed a range of marking (tattoos, brands, toe-clips) and tagging (banding, collars, PIT, VIA, VIE) methods to identify individuals and cohorts. Animals with complex life cycles are notoriously hard to mark because of the distortion or loss of the tag across metamorphosis. In amphibians, few studies have attempted larval tagging and none have been conducted on a tropical species. Here, we present the first successful account of VIE tagging in early larval stages (Gosner stage 25) of the dyeing poison frog (Dendrobates tinctorius) coupled with a novel anesthetic (2-PHE) application for tadpoles that does not require buffering. Mean weight of individuals at time of tagging was 0.12 g, which is the smallest and developmentally youngest anuran larvae tagged to date. We report 81% tag detection over the first month of development, as well as the persistence of tags across metamorphosis in this species. Cumulative tag retention vs tag observation differed by approximately 15% across larval development demonstrating that “lost” tags can be found later in development. Tagging had no effect on tadpole growth rate or survival. Successful application of VIE tags on D. tinctorius tadpoles introduces a new method that can be applied to better understand early life development and dispersal in various tropical species.

Conservation of Glomerular Organization in the Main Olfactory Bulb of Anuran Larvae.

The glomerular array in the olfactory bulb of many vertebrates is segregated into molecularly and anatomically distinct clusters linked to different olfactory functions. In anurans, glomerular clustering is so far only described in Xenopus laevis. We traced olfactory projections to the bulb in tadpoles belonging to six distantly related anuran species in four families (Pipidae, Hylidae, Bufonidae, Dendrobatidae) and found that glomerular clustering is remarkably conserved. The general bauplan consists of four unequally sized glomerular clusters with minor inter-species variation. During metamorphosis, the olfactory system undergoes extensive remodeling. Tracings in metamorphotic and juvenile Dendrobates tinctorius and Xenopus tropicalis suggest a higher degree of variation in the glomerular organization after metamorphosis is complete. Our study highlights, that the anatomical organization of glomeruli in the main olfactory bulb (MOB) is highly conserved, despite an extensive ecomorphological diversification among anuran tadpoles, which suggests underlying developmental constraints.

Neural correlates of winning and losing fights in poison frog tadpoles

Aggressive competition for resources among juveniles is documented in many species, but the neural mechanisms regulating this behavior in young animals are poorly understood. In poison frogs, increased parental care is associated with decreased water volume of tadpole pools, resource limitation, and aggression. Indeed, the tadpoles of many poison frog species will attack, kill, and cannibalize other tadpoles. We examined the neural basis of conspecific aggression in Dyeing poison frog (Dendrobates tinctorius) tadpoles by comparing individuals that won aggressive encounters, lost aggressive encounters, or did not engage in a fight. We first compared patterns of generalized neural activity using immunohistochemical detection of phosphorylated ribosomes (pS6) as a proxy for neural activation associated with behavior. We found increased neural activity in the medial pallium and preoptic area of loser tadpoles, suggesting the amphibian homologs of the mammalian hippocampus and preoptic area may facilitate loser-associated behaviors. Nonapeptides (arginine vasotocin and mesotocin) and dopamine have been linked to aggression in other vertebrates and are located in the preoptic area. We next examined neural activity specifically in nonapeptide- and tyrosine-hydroxylase-positive cells using double-label immunohistochemistry. We found increased neural activity specifically in the preoptic area nonapeptide neurons of winners, whereas we found no differences in activity of dopaminergic cells among behavioral groups. Our findings suggest the neural correlates of aggression in poison frog tadpoles are similar to neural mechanisms mediating aggression in adults and juveniles of other vertebrate taxa.

Investigating the potential use of an ionic liquid (1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide) as an anti-fungal treatment against the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

The disease chytridiomycosis, caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to global amphibian declines. Bd infects the keratinized epidermal tissue in amphibians and causes hyperkeratosis and excessive skin shedding. In individuals of susceptible species, the regulatory function of the amphibian’s skin is disrupted resulting in an electrolyte depletion, osmotic imbalance, and eventually death. Safe and effective treatments for chytridiomycosis are urgently needed to control chytrid fungal infections and stabilize populations of endangered amphibian species in captivity and in the wild. Currently, the most widely used anti-Bd treatment is itraconazole. Preparations of itraconazole formulated for amphibian use has proved effective, but treatment involves short baths over seven to ten days, a process which is logistically challenging, stressful, and causes long-term health effects. Here, we explore a novel anti-fungal therapeutic using a single application of the ionic liquid, 1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BMP-NTf2), for the treatment of chytridiomycosis. BMP-NTf2 was found be effective at killing Bd in vitro at low concentrations (1:1000 dilution). We tested BMP-NTf2 in vivo on two amphibian species, one that is relatively tolerant of chytridiomycosis (Pseudacris regilla) and one that is highly susceptible (Dendrobates tinctorius). A toxicity trial revealed a surprising interaction between Bd infection status and the impact of BMP-NTf2 on D. tinctorius survival. Uninfected D. tinctorius tolerated BMP-NTf2 (mean ± SE; 96.01 ± 9.00 μl/g), such that only 1 out of 30 frogs died following treatment (at a dose of 156.95 μL/g), whereas, a lower dose (mean ± SE; 97.45 ± 3.52 μL/g) was not tolerated by Bd-infected D. tinctorius, where 15 of 23 frogs died shortly upon BMP-NTf2 application. Those that tolerated the BMP-NTf2 application did not exhibit Bd clearance. Thus, BMP-NTf2 application, under the conditions tested here, is not a suitable option for clearing Bd infection in D. tinctorius. However, different results were obtained for P. regilla. Two topical applications of BMP-NTf2 on Bd-infected P. regilla (using a lower BMP-NTf2 dose than on D. tinctorius, mean ± SE; 9.42 ± 1.43 μL/g) reduced Bd growth, although the effect was lower than that obtained by daily doses of itracanozole (50% frogs exhibited complete clearance on day 16 vs. 100% for itracanozole). Our findings suggest that BMP-NTf2 has the potential to treat Bd infection, however the effect depends on several parameters. Further optimization of dose and schedule are needed before BMP-NTf2 can be considered as a safe and effective alternative to more conventional antifungal agents, such as itraconazole.