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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.

, the endemic Bolivian river dolphin, is threatened by anthropogenic activities including diversion of waterways for irrigation of agricultural fields, habitat degradation through deforestation, and the construction of hydroelectric dams. Within the department of Santa Cruz in Bolivia, conservation partners are committed to the capture and relocation of river dolphins that have been isolated through seasonally changing waterways, which are exacerbated by anthropogenic changes to local rivers' courses. During these rescue attempts, tissue samples were taken to better understand the genetic composition of the fragmented populations. Herein, we describe the newly sequenced and assembled Bolivian river dolphin mitochondrial genome. The genome assembly is 16,591 base pairs in length, with an overall base composition of 32.75% adenine, 25.85% thymine, 28.35% cytosine, and 13.05% guanine. This resource will pave the way for a better understanding of Bolivian river dolphin population genetics, which will help inform effective management of these vulnerable populations.

Aspergillosis is a fungal disease due to Aspergillus molds that can affect both humans and animals. As routine diagnosis remains difficult, improvement of basic knowledge with respect to its pathophysiology is critical to search for new biomarkers of infection and new therapeutic targets. Large-scale proteomics allows assessment of protein changes during various disease processes. In the present study, mass spectrometry iTRAQ® (isobaric tags for relative and absolute quantitation) protocol was used for direct identification and relative quantitation of host proteins in diseased fluids and tissues collected from an experimental rat model challenged with Aspergillus, as well as in blood obtained from naturally-infected penguins. In all, mass spectrometry analysis revealed that proteome during aspergillosis was mostly represented by proteins that usually express role in metabolic processes and biological process regulation. Ten and 17 proteins were significantly ≥4.0-fold overrepresented in blood of Aspergillus-diseased rats and penguins, respectively, while five and 39 were negatively ≥4.0-fold depleted within the same samples. In rat lungs, 33 proteins were identified with positive or negative relative changes versus controls and were quite different from those identified in the blood. Except for some zinc finger proteins, kinases, and histone transferases, and while three pathways were common (Wnt, cadherin and FGF), great inter-species variabilities were observed regarding the identity of the differentially-represented proteins. Thus, this finding confirmed how difficult it is to define a unique biomarker of infection. iTRAQ® protocol appears as a convenient proteomic tool that is greatly suited to ex vivo exploratory studies and should be considered as preliminary step before validation of new diagnostic markers and new therapeutic targets in humans.

Penguins under managed care are widely considered to have high susceptibility to infection by Aspergillus spp. Antemortem laboratory diagnostic options vary in sensitivity and specificity, and antibody detection has been problematic in penguin species given elevated levels of reactivity observed in clinically normal patients using traditional whole antigen enzyme-linked immunosorbent assays (ELISA). In the present study, an alternative assay was implemented to detect reactivity to Afmp1p, an Aspergillus cell wall antigen, in samples obtained from several different penguin species. With confirmed infection, abnormal protein electrophoretograms were consistently observed, and gliotoxin was detected in the majority of cases. An increase in reactivity to Afmp1p was observed in penguins with confirmed ( n = 18, p  < 0.0001) and probable ( n = 13, p  = 0.08) aspergillosis versus normal adult penguins ( n = 33). Interestingly, increased reactivity to Afmp1p (p < 0.0001) was noted in normal adult penguins ( n = 33) versus juvenile penguins ( n = 22, p  < 0.0001). Overall, the area under the curve for this assay was 0.890, with a sensitivity of 94.4% and a specificity of 57.6%, with an antibody assay cutoff of 1.0. Increasing reactivity resulted in an increase in specificity. These data support the use of Afmp1p antibody quantitation as part of a diagnostic workup in penguins with suspected aspergillosis.

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.

Background With 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. Methods This 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. Results Overall, 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. Conclusion Findings 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.

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.

Aspergillosis continues to be one of the most important causes of disease in captive penguins. As such, designing exhibits and holding areas that minimize the risk of aspergillosis is of great interest; however, very little has been published regarding this topic. The goal of this study was to assess total fungal spore loads as well as the loads of Aspergillus spp. encountered in multiple indoor and outdoor microenvironments around the exhibit for a large colony of African penguins (Spheniscus demersus). Air samples were collected via impaction at the microenvironments on a monthly basis over a 1-yr period. Results of this study indicated seasonal trends in both total fungal spore loads as well as Aspergillus spp. loads, with the lowest levels encountered during January through April. During the warmer, more humid spring, summer, and fall months when outdoor microenvironments experienced the highest fungal loads, the air-handling system and the pleated filters used indoors are thought to have reduced the fungal loads in the indoor microenvironments compared with the outdoor microenvironments. Additionally, surrounding planting beds were thought to contribute to the higher total fungal loads and Aspergillus spp. loads in the outdoor microenvironments. Results of this study are useful in understanding the factors that contribute to Aspergillus spp. loads in areas that house penguins, and can be used in guiding design, construction, and landscaping of penguin enclosures.

Abnormal molting, including partial or incomplete molt, arrested molt cycle, or inappropriate frequency of molt, is a primary concern for the managed African penguin (Spheniscus demersus) population and is documented across institutions. To identify factors associated with increased odds of abnormal molts and characterize intervention opportunities, a comprehensive survey evaluating numerous husbandry and medical parameters was created. Survey results represent 45 North American African penguin holding facilities and 736 unique animals. Of these individuals, 135 (18.3%) demonstrated an abnormal molt over the 5-yr study period (2012–2017). Increased odds ratios for abnormal molt included biologic (age, sex, etc.), geographic (elevation, latitude), and husbandry (exhibit design, diet, etc.) variables. The mean age of affected animals was 15.2 yr (1–45 yr, n = 135) compared with 9.92 yr (4 mo–38 yr, n = 601) for unaffected animals. In addition, although statistically insignificant, males were overrepresented in the affected cohort compared with a near even distribution among unaffected animals. Identified factors with increased odds for abnormal molting included advanced age and facilities using freshwater pools. Normally molting penguins were more commonly found with saltwater pool access and natural lighting exposure. Anecdotal medical intervention attempts are discussed, although further research is needed to define their use. Of attempted interventions, subcutaneous 5.4-mg melatonin implants placed in anticipation of environmental molting cues showed the most promise at inducing catastrophic molt, with 14 of 17 (82.3%) of affected individuals molting normally following this treatment. Survey analysis indicated that abnormal molt is a complex, multifactorial process, and modifiable factors that may predispose animals to abnormally molt exist. Addressing these factors in future exhibit design may mitigate the prevalence of this condition. Despite these efforts, it is likely that medical interventions will be required to aid in the treatment of abnormal molting in this species.

Abnormal molting, including partial or incomplete molt, arrested molt cycle, or inappropriate frequency of molt, is a primary concern for the managed African penguin (Spheniscus demersus) population and is documented across institutions. To identify factors associated with increased odds of abnormal molts and characterize intervention opportunities, a comprehensive survey evaluating numerous husbandry and medical parameters was created. Survey results represent 45 North American African penguin holding facilities and 736 unique animals. Of these individuals, 135 (18.3%) demonstrated an abnormal molt over the 5-yr study period (2012–2017). Increased odds ratios for abnormal molt included biologic (age, sex, etc.), geographic (elevation, latitude), and husbandry (exhibit design, diet, etc.) variables. The mean age of affected animals was 15.2 yr (1–45 yr, n = 135) compared with 9.92 yr (4 mo–38 yr, n = 601) for unaffected animals. In addition, although statistically insignificant, males were overrepresented in the affected cohort compared with a near even distribution among unaffected animals. Identified factors with increased odds for abnormal molting included advanced age and facilities using freshwater pools. Normally molting penguins were more commonly found with saltwater pool access and natural lighting exposure. Anecdotal medical intervention attempts are discussed, although further research is needed to define their use. Of attempted interventions, subcutaneous 5.4-mg melatonin implants placed in anticipation of environmental molting cues showed the most promise at inducing catastrophic molt, with 14 of 17 (82.3%) of affected individuals molting normally following this treatment. Survey analysis indicated that abnormal molt is a complex, multifactorial process, and modifiable factors that may predispose animals to abnormally molt exist. Addressing these factors in future exhibit design may mitigate the prevalence of this condition. Despite these efforts, it is likely that medical interventions will be required to aid in the treatment of abnormal molting in this species.