Golden Frog Research Articles

What Can Frogs Teach Us about Resilience? Adaptive Renewal in Amphibian and Academic Ecosystems.

Examples of resilience in nature give us hope amid a growing biodiversity crisis. While resilience has many definitions across disciplines, here I discuss resilience as the ability to continue to adapt and persist. Naturally, as biologists, we seek to uncover the underlying mechanisms that can help us explain the secrets of resilience across scales, from individuals to species to ecosystems and beyond. Perhaps we also ponder what the secrets to resilience are in our own lives, in our own research practices, and academic communities. In this paper, I highlight insights gained through studies of amphibian resilience following a global disease outbreak to uncover shared patterns and processes linked to resilience across amphibian communities. I also reflect on how classical resilience heuristics could be more broadly applied to these processes and to our own academic communities. Focusing on the amphibian systems that I have worked in-the Golden Frogs of Panama (Atelopus zeteki/varius) and the Mountain Yellow-Legged Frogs of California (Rana muscosa/sierrae)-I highlight shared and unique characteristics of resilience across scales and systems and discuss how these relate to adaptive renewal cycles. Reflecting on this work and previous resilience scholarship, I also offer my own thoughts about academia and consider what lessons we could take from mapping our own adaptive trajectories and addressing threats to our own community resilience.

CASE DEFINITION AND TREATMENT TRIAL OF TETANY SYNDROME IN PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI).

The mass extinction of amphibians necessitates specialized programs to ensure species' survival. Maryland Zoo in Baltimore houses the largest assurance population of the critically endangered Panamanian golden frog (Atelopus zeteki). However, individuals in this population experience a tetany-like syndrome, characterized by rigid/inappropriately positioned limbs and difficulty hopping, swimming, and righting. In this study, a syndrome case definition was assigned and the associated clinical signs were described. Then, four different treatments were systematically assessed in order to find the most effective protocol for treatment and begin to elucidate its underlying causes. Eighty-three frogs fulfilled the case definition and were treated orally for 14 d with either calcium gluconate, magnesium chloride, supplemental gavage feeding, or combination of calcium, magnesium, and vitamin B complex. Frogs were tested with a defined protocol assessing hopping, righting, and swimming abilities. Testing was performed at symptom onset and repeated weekly until resolution occurred. Analyses revealed that combination treatment was significantly more effective in eliminating clinical signs of tetany syndrome. Results show the most effective way to treat this syndrome, but do not help elucidate the underlying cause. Future work will focus on examining factors (e.g., diet, husbandry) that may elicit the syndrome for a more complete understanding of its etiology.

PREVALENCE OF OCULAR ABNORMALITIES AND NORMAL DIAGNOSTIC PARAMETERS IN TWO GROUPS OF PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI).

Panamanian golden frog (PGF) (Atelopus zeteki) is a critically endangered species. The Maryland Zoo in Baltimore houses two groups of PGF originating from distinct geographic locations as an assurance colony, with the goal of upholding genetics for future release of individuals back to their native environment. The purpose of this cross-sectional study was to characterize the prevalence of ocular abnormalities in these two zoo-housed populations of PGF as well as to establish normal parameters for selected diagnostic tests in these groups. Twenty-five females and 25 males were randomly selected from each group (100 PGF; 200 eyes in total) to undergo ocular examination using slit lamp biomicroscopy and direct ophthalmoscopy. Endodontic absorbent paper point test (EAPPT) and intraocular pressure (IOP) and Rose Bengal stain diagnostic tests were also performed. Reference ranges for tear production (EAPPT, 0.5-3 mm/min) and IOP (14-26 mmHg) were calculated in the nondiseased PGF eyes (n = 160 eyes). Rose Bengal stain uptake was negative on all eyes. In total, 40 eyes of 30 PGF were found to have some form of ocular abnormality (28% of PGF, 20% of eyes). The most frequently observed ocular abnormalities were cataract (9% of PGF, 6% of eyes) and keratitis (nonlipid keratopathy; 10% of PGF, 5.5% of eyes). There was no significant difference in overall ocular abnormality prevalence between the two groups studied (P = 0.37) or between the sexes (P = 0.76). The median age of an eye with cataract and keratitis (nonlipid keratopathy) was 10.35 and 7.7 yr, respectively. Ocular abnormalities are common in these two populations of PGF. Documentation of these ocular abnormalities and establishment of diagnostic reference ranges have not previously been published and may be important for maintaining the health of this endangered species.

Critical calls: Circadian and seasonal periodicity in vocal activity in a breeding colony of Panamanian golden frogs (Atelopus zeteki).

The Panamanian golden frog (Atelopus zeteki) is a critically endangered species and currently is believed to survive and reproduce only in human care. Panamanian golden frog males are considerably vocal which may be an important component in their successful reproduction, though little is currently known about their calls. To better understand the behavior and vocal patterns of this species and to improve breeding efforts in the assurance colony, we employed individual sound recording of male advertisement calls and acoustic monitoring of a breeding colony to investigate variation in the vocal behavior of Panamanian golden frogs. The goal was to capture variability within and among frogs as well as patterns of periodicity over time. First, the advertisement calls from individual male Panamanian golden frogs were recorded, and acoustic parameters were analyzed for individual differences. Results suggest that male advertisement calls demonstrate individual- and population specificity. Second, data collected through a year-long acoustic monitoring of the breeding colony were investigated for circadian and circannual periodicity. Male vocal activity revealed a circadian periodicity entrained by the daily light schedule. Seasonal periodicity was also found with highest vocal activities between December and March. The finding of a seasonal periodicity is worth noting given that the population had been bred for 20 years under constant environmental conditions. Finally, results suggest that vocal activity was responsive to daily animal care activity. Vocal activity decreased substantially when personnel entered the room and engaged in animal husbandry activities. The findings illustrate the usefulness of acoustic monitoring to provide insight into animal behavior in a zoo setting in a key breeding colony of endangered animals, and calling pattern observations may be utilized to modify husbandry practices to improve Panamanian golden frog breeding success and general care.

High relative humidity and temperature limit disease development and mortality in golden frogs of Panama, Atelopus zeteki, infected with Batrachochytrium dendrobatidis

AbstractTo combat the loss of species due to emerging infectious diseases, scientists must incorporate ecological parameters, such as temperature and humidity, to understand how the environment affects host–pathogen interactions. The fungal disease chytridiomycosis is a compelling case study to investigate the role of both temperature and humidity on infectious disease, as both the fungal pathogen (Batrachochytrium dendrobatidis, Bd) and the host (amphibians) are heavily influenced by these abiotic factors. We performed two experiments to investigate the importance of relative humidity and temperature on frog immunity (production of antimicrobial skin secretions) and disease development in captive golden frogs (Atelopus zeteki) of Panama. We found that the quantity of skin secretions significantly decreased over time in frogs moved from low to medium and high relative humidity treatments. Following Bd exposure, frogs in high temperature (26–27 °C) and high relative humidity (80–90%) had lower pathogen loads and survived significantly longer than frogs kept in all other treatment conditions, including high temperature and low relative humidity. These results suggest that high relative humidity may be an important, although less understood, mediator of Bd infection and the survival of golden frogs. Because the environment can drastically alter disease dynamics, understanding how temperature and humidity influence chytridiomycosis outcomes in golden frogs may be essential for the success of the reintroduction of captive frogs.

Count. By Valerie Martínez.

Valerie Martínez’s most recent book of poems, published by the Camino del Sol Latinx Literary Series, begins with what is described as “a critical drought”: a soapy, wet carwash staffed by teenage girls “in cutoffs and bikinis” and advertised as “CARWASH U NO U WANT ONE” (1). Martínez’s response to this scene of drought, both critical and literal, is a flood of stories, those that recount both deluge and survival. Running alongside each other, these narratives weave in and out of the book-length poem: a spectral girl approaching and retreating from the ocean; a realist retelling of the speaker’s 53-day hike along the Santa Fe, “a dying river”; and Indigenous accounts of how humans may be able learn, through disaster, to live in balance. Together, they register wave after wave of what we’ve lost, and what we stand to lose. But while the poem is an elegy for the endangered and the extinct—golden frogs and staghorn coral, mondo grass at the end of its ability to adapt—Count also resists a familiar ecopoetic despair in its varied responses to environmental injustice and threat. From outraged silence—“I stifle the urge to mumble and cough” (10)—to helpless inaction—“All he could do: watch” (24) to an almost-acceptance—“we’ve had/our short time—brilliant and careless” (37), the speaker witnesses the world multiply with “wonder, grief,/urgency”(22). And while false comfort is rejected—such comfort is “[a] ruse, palliation”(25)—on the other side of this flood of loss are the stories of survival and regeneration: the ice plant closing its stomata to preserve water; a crab remaking its gills in response to changing conditions; the repetition of scant but real human survival in the Indigenous stories that resist and rewrite settler and capital-driven narratives. At the close of the book, the poem offers up a particularly powerful way to redress the losses incurred by peoples consistently “at odds with the earth” (41) in the story of Hadanisht’é, a deity who puts humans to work reassembling “a disintegrating world,” then teaches them to live with more grace on a planet both fractured and beautiful (43).

When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis.

To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

Influence of oviposition-inducing hormone on spawning and mortality in the endangered Panamanian golden frog (Atelopus zeteki)

BackgroundWith Panamanian golden frogs (Atelopus zeteki; PGFs) likely extirpated from the wild, ensuring long-term sustainability of captive populations is crucial in order to conserve this critically endangered species. Unfortunately, PGFs display a unique reproductive behavior involving a prolonged period of amplexus leading to challenges in their successful captive propagation. The Maryland Zoo in Baltimore has observed high levels of mortality during the breeding season and suboptimal reproductive success leading to the use of hormone stimulation to aid in reproduction and health management.MethodsThis project aimed to develop induced ovulation and health management protocols by (1) evaluating different doses of gonadotropin releasing hormone analogue (GnRHa), (2) comparing the efficacy of GnRHa and GnRHa + metoclopramide, (3) determining latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Female PGFs (n = 174) were given GnRHa either in various concentrations (Experiment 1) or combined with metoclopramide (Experiment 2), and oviposition success, latency, and mortality were measured as binary response variables.ResultsOverall, the use of exogenous hormones significantly decreased mortality when compared to the control data of natural egg-laying females. GnRHa doses of 0.05 μg/g body weight produced similar ovulation rates compared to higher doses, and the addition of metoclopramide did not increase oviposition success compared to GnRHa alone. Lastly, results indicate the majority of female PGFs will release eggs within 48 h following the initial pulse of hormones with a small percentage ovipositing after a second pulse.ConclusionFindings from this study will benefit captive management of PGFs by documenting the increased survival of females when given hormone stimulation and defining appropriate GnRHa doses and expected latency to spawning.

Long-term urbanization impacts the eastern golden frog (Pelophylax plancyi) in Shanghai City: Demographic history, genetic structure, and implications for amphibian conservation in intensively urbanizing environments.

Understanding the mechanisms of how urbanization influences the evolution of native species is vital for urban wildlife ecology and conservation in the Anthropocene. With thousands of years of agriculture‐dominated historical urbanization followed by 40 years of intensive and rapid urbanization, Shanghai provides an ideal environment to study how the two‐stage urbanization process influences the evolution of indigenous wildlife, especially of anuran species. Therefore, in this study, we used mitochondrial Cyt‐b gene, microsatellite (SSR), and single nucleotide polymorphism (SNP) data to evaluate the demographic history and genetic structure of the eastern golden frog (Pelophylax plancyi), by sampling 407 individuals from 15 local populations across Shanghai, China. All local populations experienced bottlenecks during historical urbanization, while the local populations in urban areas maintained comparable contemporary effective population sizes (N e) and genetic diversity with suburban and rural populations. Nevertheless, the rapid modern urbanization has already imposed significant negative effects to the integrity of populations. The 15 local populations were differentiated into eight genetic clusters, showing a spatial distribution pattern consistent with the current urbanization gradient and island–mainland geography. Although moderate gene flow still occurred from the rural peripheral cluster to urban and suburban clusters, population fragmentation was more serious in the urban and suburban populations, where higher urbanization levels within 2‐km radius areas showed significant negative relationships to the N e and genetic diversity of local populations. Therefore, to protect urban wildlife with limited dispersal ability, improving conditions in fragmented habitat remnants might be most essential for local populations living in more urbanized areas. Meanwhile, we highlight the need to preserve large unfragmented rural habitats and to construct corridor networks to connect discrete urban habitat remnants for the long‐term wildlife conservation in intensively urbanizing environments.

Givaudan buying another naturals firm

Givaudan is purchasing Ungerer, a New Jersey–based company that produces essential oils for flavors and fragrances, for an undisclosed sum. Ungerer is a 125-year-old, family-owned company with facilities in the US, UK, Mexico, Colombia, Uruguay, India, China, and Australia. Givaudan and its rivals Firmenich and International Flavors & Fragrances have been buying smaller firms to increase their access to naturally derived ingredients. Since 2018, Givaudan has bought Expressions Parfumées, Albert Vieille, and Vietnam’s Golden Frog.

Fighting a fungal scourge

Researchers trying to rescue amphibians from a global fungus epidemic are finding that bacteria may be their best allies . More than a decade ago, amphibian microbial ecologist Reid Harris watched a mother salamander as she marched in a figure-eight pattern through her clutch of soft, jellylike eggs. He knew that her strange walk, rubbing up against her brood, transferred beneficial bacteria from her skin onto the eggs to fight off fungal infection. Then something clicked. Harris wondered if she might also be showing him the solution to a scourge threatening hundreds of other amphibian species around the world. To fight the deadliest wildlife disease on record, a fungus responsible for the decline or disappearance of hundreds of amphibian species including the Panamanian golden frog, researchers are looking to the bacteria that live on frog skin. Image credit: Flickr/Brian Gratwicke. Then, as now, amphibians were dying in droves. At least one-third of species face extinction, with at least 90 confirmed gone and another 500 in decline (1, 2). The major culprit is the skin disease chytridiomycosis, caused by the waterborne fungus Batrachochytrium dendrobatidis , or Bd for short. Under a microscope, fungal bodies anchor themselves in the translucence of frog skin and often group together to form a collection of small spheres. Chytridiomycosis kills by disrupting ion and fluid transport, eventually stopping the host’s heart (3). A particularly deadly Bd lineage arose in the Korean peninsula sometime in the last century, the source of a pandemic that fanned out across the world through the global amphibian trade, infecting about 700 species in six global regions over the past five decades (4). Naive immune systems (mostly those of frogs) couldn’t fight off the highly contagious and aggressive pathogen, and chytridiomycosis spread like a leveling wave, becoming the most devastating …

Business Roundup

BUSINESS Business Roundup ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2019, 97 (21), p 11May 27, 2019Cite this:C&EN 97, 21, 11AbstractBASF plans to build engineering plastics and thermoplastic polyurethane plants at the big petrochemical complex it is planning for Zhangjiang, China. BASF says the facilities will open in 2022 and be the first production plants at the new site. Amyris, a developer of chemicals made from sugar, has received its first $10 million milestone payment from Lavvan for progress on a fermentation pathway for cannabinoids. Lavvan says its first cannabinoid product, set to launch next year, will be higher purity and lower cost than similar products. Momentive’s sale to a consortium of South Korean companies has been completed. The deal for the silicones maker was valued at $3.1 billion. Shell Chemical has selected its complex in Geismar, Louisiana, as the site for a planned ethylene glycol plant. The company is still conducting feasibility studies for the plant, which is expected to cost $1.2 billion. Givaudan has acquired Golden Frog, aView: PDF | Full Text HTML

Ophthalmic findings in a captive population of Panamanian golden frogs: Atelopus zeteki.

The aim of this study was to describe normal ocular morphology and establish parameters for select diagnostics in a population of Panamanian Golden Frogs (PGF) Atelopus zeteki maintained at the Detroit Zoo. A total of 22 toads free of ocular disease were included in this study. Ophthalmic diagnostic tests included as follows: endodontic absorbent paper point tear test (EAPPTT), assessment of intraocular pressures (IOP), Rose Bengal stain, palpebral fissure width, blink rate and corneal cultures. Histology was performed on stored postmortem samples. Panamanian Golden Frogs have a distinct brow ridge and mobile third eyelid. Biomicroscopy revealed avascular, convex corneas with a shallow anterior chamber. Iris color ranged from yellow to green with dark pigmentation of the peripheral iris. Pupil shape was round. Posterior segment and fundus could not be visualized. Total number of blinks observed over a 10-minute period was one. Mean EAPPTT was 2±1mm and 3±1mm in males and females, respectively (P). Mean IOP using rebound tonometry in horizontal and vertical restraint positions were 21.6±2.28mmHg and 17.45±3.66mmHg, 21.85±2.7mmHg and 17.5±3.93mmHg in males and females, respectively. Mean palpebral fissure width was 5.02±0.46mm and 6.12±0.39mm in males and females, respectively (P). Enterococcus faecalis was isolated from one clinically normal toad. Tear production and blink rate were low and not significantly different between males and females. Intraocular pressure was significantly greater in male toads (P value <0.01). Females had a significantly larger palpebral fissure width when compared to males (P value <0.01) 5819. The ophthalmic parameters established in this study can aid in the evaluation and diagnosis of eye diseases in PGF.

Wittgenstein

Wittgenstein Andre Bagoo (bio) They bought Wittgenstein an outfit to wear on chilly nights. Both men felt he liked it so much (he jumped wildly when presented with the unicorn-patterned onesie) they went back to the pet shop for seven more: one for each day of the week plus a spare for laundry days. They took pictures of him in his pajamas for his Instagram, tagging the pet shop. In all the posts, he stared at the camera. He was a black and white mongrel, between a husky and a pariah, very foxlike in the face, the kind of dog you might see running up and down the pavements of the city looking uneasily from side to side wondering where its next meal was coming from, except he had been rescued by them. On nights when Enzo was at work, James would take pictures of him tucked into his dog bed with blankets and cushions and send these pictures to Enzo, saying GOODNIGHT XO. On nights when Enzo was not at work, they would tuck Wittgenstein in together, a process that involved cuing up and singing "So Long Farewell" from The Sound of Music. They called the part sung by the youngest von Trapp, Gretl, "Wittgenstein's part." In the new apartment, the location of which they chose solely because of him (the landlord was pet-friendly; it was close to several parks and pet shops), they developed a morning routine with Wittgenstein taking his place in the breakfast nook, being fed bits of sweetbread and ham. When Enzo left for work, he would tell Wittgenstein he was now "in charge," while James would open the window overlooking the plant-lined driveway so Wittgenstein could position himself to watch. As he drove past the window, Enzo would cry "Wittgenstein! Wittgenstein! Wittgenstein!" and the pet would start twirling like a hurricane. They decorated the apartment sparingly, opting to match the existing palette (which happened to suit Wittgenstein's fur). They went to Mode Alive on Frederick Street in Port of Spain and picked out large wooden signs coated in chipped paint. One said LOVE IS ALL YOU NEED; another DOGS WELCOME, PEOPLE TOLERATED; and another TO ACCOMPLISH GREAT THINGS WE MUST NOT ONLY ACT, BUT ALSO DREAM; NOT ONLY PLAN, BUT ALSO BELIEVE. They briefly worried about whether people would think these signs corny, then dismissed such thoughts. Enzo felt having textual elements in the décor matched the fact that James was a writer, while James liked the way the signs were distressed, reminding him of the country house he'd spent August vacations in as a child — a rustic place he associated with love, the kind of place he hoped they might one day retire to together, spending days reading classic West Indian novels under mango trees and nights drinking wine under the starlight amid wildflowers and small golden frogs. They carried their haul to their truck lower down the street, strolling through the shadowless heat of the city. When they got home, James searched the internet and discovered the [End Page 101] sign about dreaming and believing was a quote from a once-famous French writer, and it seemed the sign was meant to hang over his writing desk, he was meant to be a writer, and they were, all three of them, meant to be together in this new place which they had moved into after only knowing each other for six months because James had been evicted from his old place and they both liked the idea of raising Wittgenstein together. They didn't think it too risky to move in so soon. This was how things had worked out. It was meant to be. In the initial months, the apartment filled with music. They blasted "Dog Years" by Maggie Rogers. They played "Dog Days Are Over" by Florence and the Machine (though observing that their dog days were, luckily, not over). When they went to a yard sale on Alfredo Street, they found old CDs. They picked out Hounds of Love by Kate Bush and began playing that on loop to the chagrin of their neighbors Frank and Betsy, who often glimpsed them frolicking...

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth.

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host's skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.

CAUSES OF MORTALITY IN CAPTIVE PANAMANIAN GOLDEN FROGS ( ATELOPUS ZETEKI) AT THE MARYLAND ZOO IN BALTIMORE, 2001-2013.

The Maryland Zoo in Baltimore is home to the largest captive assurance population of the critically endangered Panamanian golden frog ( Atelopus zeteki). With the ongoing extinction that is occurring worldwide in amphibians, the need for amphibian captive assurance populations is growing, and few mortality reviews on amphibian species exist. Necropsy and histopathologic examination of animals that die in captivity can help identify population-level disease problems, direct research needs in amphibian medicine and husbandry, and improve the success of captive breeding programs. This study reviews postmortem findings from 406 frogs, greater than 1 yr of age, which died in this population from 2001 to 2013. Frogs were categorized by age and sex, and the cause of mortality was determined. Dermatitis associated with filamentous-type fungal organisms was the most common cause of mortality in both age and sex categories and accounted for one-third of frog deaths in this study (36.0%; n = 146 out of 406 frogs). Other major causes of mortality included renal disease, gastrointestinal disease, septicemia, and a previously undescribed myopathy condition associated with a tetany syndrome. Increased mortality of frogs occurred during the breeding season, highlighting the need for further research into methods to minimize mortality during this time.

Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki)

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9 GnRH; 4 μg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (<50%) as well as DNA integrity (32.8% of intact cells) after 23 min while morphology did not differ (30.4% of normal cells), regardless of the hormone stimulation. This is the first systematic study on the effect of extracellular environment over time on A. zeteki sperm quality. This will contribute to the development of sperm handling protocols and reproductive technologies for this and other endangered Atelopus species.

THE PHARMACOKINETICS OF TOPICAL ITRACONAZOLE IN PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI).

Chytridiomycosis is caused by the fungus Batrachochytrium dendrobatidis and is one of the primary causes of the global decline in amphibian populations and specifically of the Panamanian golden frog ( Atelopus zeteki ). Itraconazole has been demonstrated to be an effective treatment for chytridiomycosis by inhibiting cytochrome P450, a major enzyme important for the structure of B. dendrobatidis zoospores' plasma membranes. However, anecdotal reports of toxicity in this and other amphibian species have been reported at the 0.01% concentration. This study is the first to determine pharmacokinetics of 0.01% and 0.001% itraconazole in the Panamanian golden frog. Frogs were bathed 10 min, euthanized, and skin, liver, and heart were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 36 hr. Itraconazole concentrations were measured using high performance liquid chromatography, and the minimum inhibitory concentration (MIC) of itraconazole (0.032 μg/ml) for B. dendrobatidis was used to determine whether therapeutic concentrations were attained. Itraconazole was detected in all tissues at both concentrations, indicating systemic absorption. At the 0.01% itraconazole bath, itraconazole concentrations in all tissues exceeded the MIC at all time points, and the lack of decline until the end of the study at 36 hr precluded determining a disappearance half-life. With the 0.001% bath, itraconazole exceeded the MIC and declined with a disappearance half-life that markedly varied (14.1-1,244 min). This study augments the growing literature base on chytridiomycosis and seeks to aid in further experimental attempts to find the most-optimal treatment protocol for this disease.

Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki)

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9--GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 106 cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 106 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7–6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50–70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.