Eria A. Rebollar

Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis

Symbiotic bacteria on amphibian skin can inhibit growth of the fungus Batrachochytrium dendrobatidis (Bd) that has caused dramatic population declines and extinctions of amphibians in the Neotropics. It remains unclear how the amphibians’ skin microbiota is influenced by environmental bacterial reservoirs, host-associated factors such as susceptibility to pathogens, and pathogen presence in tropical amphibians. We sampled skin bacteria from five co-occurring frog species that differ in Bd susceptibility at one Bd-naive site, and sampled one of the non-susceptible species from Bd-endemic and Bd-naive sites in Panama. We hypothesized that skin bacterial communities (1) would be distinct from the surrounding environment regardless of the host habitat, (2) would differ between Bd susceptible and non-susceptible species and (3) would differ on hosts in Bd-naive and Bd-endemic sites. We found that skin bacterial communities were enriched in bacterial taxa that had low relative abundances in the environment. Non-susceptible species had very similar skin bacterial communities that were enriched in particular taxa such as the genera Pseudomonas and Acinetobacter. Bacterial communities of Craugastor fitzingeri in Bd-endemic sites were less diverse than in the naive site, and differences in community structure across sites were explained by changes in relative abundance of specific bacterial taxa. Our results indicate that skin microbial structure was associated with host susceptibility to Bd and might be associated to the history of Bd presence at different sites.

More than skin deep: functional genomic basis for resistance to amphibian chytridiomycosis.

The amphibian-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is one of the most generalist pathogens known, capable of infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is commonly evoked for these resistant or tolerant species, yet to date, we have no direct comparison of amphibian species responses to infection at the level of gene expression. In this study, we challenged four Central American frog species that vary in Bd susceptibility, with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential expression tests and coexpression gene network analyses. We found that resistant species have reduced skin inflammatory responses and increased expression of genes involved in skin integrity. In contrast, only highly susceptible species exhibited suppression of splenic T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.

Using "Omics" and Integrated Multi-Omics Approaches to Guide Probiotic Selection to Mitigate Chytridiomycosis and Other Emerging Infectious Diseases

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called “omics,” are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov–Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.

Panamanian frog species host unique skin bacterial communities

Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.

Role of the p53 homologue from Drosophila melanogaster in the maintenance of histone H3 acetylation and response to UV-light irradiation

It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. In this work we explored if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, we analyzed K9 and K14 H3 acetylation patterns in situ after UV irradiation of wild‐type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild‐type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3‐K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3‐K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans.

The Lethal Fungus Batrachochytrium dendrobatidis Is Present in Lowland Tropical Forests of Far Eastern Panamá

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15–34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11–24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen.

Water-sediment niche differentiation in ancient marine lineages of Exiguobacterium endemic to the Cuatro Cienegas Basin.

The evolutionary history and ecological differentiation of the genus Exiguobacterium was characterized within natural communities from the Cuatro Cienegas Basin, Mexico. Exiguobacterium comprises both halophilic and alkaliphilic bacteria that are abundant among the aquatic systems of the Cuatro Cienegas Basin. We obtained complete sequences of the 16srRNA gene and partial sequences of four housekeeping genes (citC, rpoB, recA and hsp70) in 183 Exiguobacterium isolates retrieved from distinct aquatic systems. We defined three main phylogroups that are closely related to marine and thermophilic species of the genus. These phylogroups were neither specific to a given aquatic system nor to a particular salinity. Phylogenetic reconstruction indicated the presence of several small clusters within the phylogroups. These clusters consisted of isolates predominantly retrieved from sediment or water. Unifrac and AdaptML analyses confirmed this observation, pointing towards a clear pattern of differentiation linked to either sediment or water habitats. Our results are in line with the concept that niche differentiation is one of the main factors shaping prokaryotic populations and leading to evolutionary divergence.

An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression

Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5′ non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF.

Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.

Population expansions shared among coexisting bacterial lineages are revealed by genetic evidence

Comparative population studies can help elucidate the influence of historical events upon current patterns of biodiversity among taxa that coexist in a given geographic area. In particular, comparative assessments derived from population genetics and coalescent theory have been used to investigate population dynamics of bacterial pathogens in order to understand disease epidemics. In contrast, and despite the ecological relevance of non-host associated and naturally occurring bacteria, there is little understanding of the processes determining their diversity. Here we analyzed the patterns of genetic diversity in coexisting populations of three genera of bacteria (Bacillus, Exiguobacterium, and Pseudomonas) that are abundant in the aquatic systems of the Cuatro Cienegas Basin, Mexico. We tested the hypothesis that a common habitat leaves a signature upon the genetic variation present in bacterial populations, independent of phylogenetic relationships. We used multilocus markers to assess genetic diversity and (1) performed comparative phylogenetic analyses, (2) described the genetic structure of bacterial populations, (3) calculated descriptive parameters of genetic diversity, (4) performed neutrality tests, and (5) conducted coalescent-based historical reconstructions. Our results show a trend of synchronic expansions across most populations independent of both lineage and sampling site. Thus, we provide empirical evidence supporting the analysis of coexisting bacterial lineages in natural environments to advance our understanding of bacterial evolution beyond medical or health-related microbes.