Nonlethal amphibian skin swabbing of cutaneous natural products for HPLC fingerprinting

Umile, Thomas P.; McLaughlin, Patrick J.; Johnson, Kendall R.; Honarvar, Shaya; Blackman, Alison L; Burzynski, Elizabeth A.; Davis, Robert W.; Teotonio, Thais L.; Hearn, Gail W.; Hughey, Christine A.; Lagalante, Anthony F.; Minbiole, Kevin P. C.

doi:10.1039/c4ay00566j

Cited by 19 publications

(13 citation statements)

References 17 publications

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“…B 282: 20142881 infection further supports the hypothesis that either microbial or host-produced metabolites influenced infection dynamics. Our methods for chemical analysis (reverse-phase LCMS) have been optimized for observation of bacterial metabolites [36]. Many known metabolites of amphibian origin that inhibit pathogens and provide defence against predators, such as antimicrobial peptides [62] and zetekitoxin [63], respectively, are too polar to be observed in our conditions.…”

Section: Discussionmentioning

confidence: 99%

“…The combined crude extracts were filtered (0.45 mm PTFE membrane), concentrated and reconstituted in methanol. Samples were analysed with high performance liquid chromatography-mass spectrometry (LCMS) following Umile et al [36]. To detect intersample contamination, we inserted methanol injections into the LCMS queue after every five samples.…”

Section: (E) Extraction Of Cutaneous Metabolites and Lcmsmentioning

confidence: 99%

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Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

et al. 2015

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Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.

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Section: Discussionmentioning

confidence: 99%

Section: (E) Extraction Of Cutaneous Metabolites and Lcmsmentioning

confidence: 99%

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

et al. 2015

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“…We sampled the cutaneous bacterial community using sterile rayon swabs (MW113; Medical Wire Equipment & Co. Ltd., Corsham, United Kingdom) as in Walke et al (29). We then used foam-tipped swabs to sample metabolite profiles as in Umile et al (34). Prior to use, the polyurethane swabs were prerinsed twice in methanol to remove methanol-soluble impurities and were then allowed to dry under sterile conditions in the laboratory.…”

Section: Methodsmentioning

confidence: 99%

“…For metabolite analysis, we used methods similar to Umile et al (34). To extract metabolites from the foam swabs, 1.0 ml of methanol was added to each swab tip in its centrifuge tube.…”

Section: Methodsmentioning

confidence: 99%

Short-Term Exposure to Coal Combustion Waste Has Little Impact on the Skin Microbiome of Adult Spring Peepers (Pseudacris crucifer)

Hughey

Walke

Becker

et al. 2016

Appl Environ Microbiol

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Disruptions to the microbiome can impact host health as can exposure to environmental contaminants. However, few studies have addressed how environmental contaminants impact the microbiome. We explored this question for frogs that breed in wetlands contaminated with fly ash, a by-product of coal combustion that is enriched in trace elements. We found differences in the bacterial communities among a fly ash-contaminated site and several reference wetlands. We then experimentally assessed the impacts of fly ash on the skin microbiome of adult spring peepers (Pseudacris crucifer). Frogs were exposed to fly ash in the laboratory for 12 h, the duration of a typical breeding event, and the skin microbiome was assessed after 5 days (experiment 1) or after 5 and 15 days (experiment 2). We examined bacterial community structure using 16S rRNA gene amplicon sequencing and metabolite profiles using high-pressure liquid chromatography-mass spectrometry (HPLC-MS). We found little impact as the result of acute exposure to fly ash on the bacterial communities or metabolite profiles in either experiment, suggesting that the bacterial symbiont communities of adults may be relatively resistant to brief contaminant exposure. However, housing frogs in the laboratory altered bacterial community structure in the two experiments, which supports prior research suggesting that environmental source pools are important for maintaining the amphibian skin microbiome. Therefore, for contaminants like fly ash that may alter the potential source pool of symbionts, we think it may be important to explore how contaminants affect the initial assembly of the amphibian skin microbiome in larval amphibians that develop within contaminated sites. IMPORTANCEAnimals are hosts to many symbiotic microorganisms, collectively called the microbiome, that play critical roles in host health. Therefore, environmental contaminants that alter the microbiome may impact hosts. Some of the most widespread contaminants, produced worldwide, are derived from the mining, storage, and combustion of coal for energy. Fly ash, for example, is a by-product of coal combustion. It contains compounds such as arsenic, selenium, cadmium, and strontium and is a recognized source of ground and surface water contamination. Here, we experimentally investigated the impacts of short-term fly ash exposure on the skin microbiome of spring peepers, one of many species of amphibian that sometimes breed in open fly ash disposal ponds. This research provides a look into the potential impacts of fly ash on an animal's microbiome and suggests important future directions for research on the effects of environmental contaminants on the microbiome. Vertebrates host a wide array of symbiotic microorganisms, mainly in the gut, but there are unique microbial communities spread throughout the body from the nasal cavity to the lungs to the skin (1). While we have long understood that the microbiota residing in the gut are diverse and are important in helping to digest food, only recently have...

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“…Biomedical applications include continued development of high-performance thin-layer chromatography (HPTLC) for pharmaceutical and tissue sample analysis, 43,44 a capillary electrophoresis method to detect hemoglobin polymers in blood for anti-doping tests, 45 and a demonstration that iontophoresis in tissue may include an electroosmotic component. 47 For analysis of complex mixtures, such as polyaromatic hydrocarbons in urine, one PUI research group demonstrated the advantages of twodimensional gas chromatography. 47 For analysis of complex mixtures, such as polyaromatic hydrocarbons in urine, one PUI research group demonstrated the advantages of twodimensional gas chromatography.…”

Section: Separationsmentioning

confidence: 99%

Analytical chemistry research at primarily undergraduate institutions: training tomorrow's investigators

Kovarik

2015

Anal. Methods

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Primarily undergraduate institutions (PUIs) are a unique training ground for young scientists who work closely with faculty on their research. Researchers at PUIs face a number of challenges, including balancing research and teaching responsibilities, obtaining sufficient funding for projects, and training students in their first research experiences. Faculty at PUIs deploy a variety of strategies to address these challenges, and the resulting research provides important benefits to faculty, students, the analytical chemistry community, and society. This perspective discusses these challenges and benefits in detail.Additionally, several vignettes describe how specific faculty members have established and maintained productive research programs at PUIs, and select publications since 2009 by PUI researchers are highlighted.

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Nonlethal amphibian skin swabbing of cutaneous natural products for HPLC fingerprinting

Cited by 19 publications

References 17 publications

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Short-Term Exposure to Coal Combustion Waste Has Little Impact on the Skin Microbiome of Adult Spring Peepers (Pseudacris crucifer)

Analytical chemistry research at primarily undergraduate institutions: training tomorrow's investigators

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