Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health. Microorganisms represent a large portion of global biodiversity and are responsible for many important ecological functions, yet the majority of microbes have not been cultured (1-3). It is estimated that only 0.001% to 15% of microbes can be cultured by use of commonly used techniques and media (2, 4), although some recent attempts at culturing the uncultured have been successful. For example, a prevalent and abundant human gut bacterium with potential biomedical importance was recently cultured by a gene-targeted approach (5). Still, only a third of known bacterial phyla have cultured representatives (6). Recent advances in culture-independent rRNA-based molecular approaches have allowed for a greater understanding of bacterial diversity (1,7,8), but in most cases, the only data we have about uncultured microbes are the DNA sequences obtained directly from the environment. Next-generation sequencing in some cases has led to the discovery of entire groups, such as the TM7 candidate division, often found in terrestrial, aquatic, and clinical habitats (9). This group is phylogenetically distinct and widespread yet has not been isolated in pure culture (9-12). There are also bacteri...

One current challenge in microbial ecology is elucidating the functional roles of the large diversity of free-living and host-associated bacteria identified by culture-independent molecular methods. Importantly, the characterization of this immense bacterial diversity will likely require merging data from culture-independent approaches with work on bacterial isolates in culture. Amphibian skin bacterial communities have become a recent focus of work in host-associated microbial systems due to the potential role of these skin bacteria in host defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd), which is associated with global amphibian population declines and extinctions. As there is evidence that some skin bacteria may inhibit growth of Bd and prevent infection in some cases, there is interest in using these bacteria as probiotic therapy for conservation of at-risk amphibians. In this study, we used skin swabs from American toads (Anaxyrus americanus) to: (1) assess the diversity and community structure of culturable amphibian skin bacteria grown on high and low nutrient culture media, (2) determine which culture media recover the highest proportion of the total skin bacterial community of individual toads relative to culture-independent data, and (3) assess whether the plated communities from the distinct media types vary in their ability to inhibit Bd growth in in-vitro assays. Overall, we found that culture media with low nutrient concentrations facilitated the growth of more diverse bacterial taxa and grew distinct communities relative to media with higher nutrient concentrations. Use of low nutrient media also resulted in culturing proportionally more of the bacterial diversity on individual toads relative to the overall community defined using culture-independent methods. However, while there were differences in diversity among media types, the variation among individual hosts was greater than variation among media types, suggesting that swabbing more individuals in a population is the best way to maximize culture collections, regardless of media type. Lastly, the function of the plated communities against Bd did not vary across culture media type or between high and low nutrient media. These results inform current efforts for developing a probiotic-based approach for amphibian conservation and help to ensure that culture collections are capturing the majority of the important diversity in these systems.

Some amphibian skin bacteria inhibit growth of a fungal amphibian pathogen, Batrachochytrium dendrobatidis (Bd), but it is unclear how dominant these anti-Bd bacteria are in skin communities. Using in vitro co-culture challenge assays, we quantified Bd inhibition by bacterial isolates collected from the skin of four amphibian species: bullfrogs, Eastern newts, spring peepers and American toads. The 16S rRNA sequences for each isolate were matched to culture-independent amplicon sequences from the same individuals to assess inhibitory function versus relative abundance. Dominant bacteria had higher Bd inhibition than rare bacteria in bullfrog and newt populations, in which Bd was prevalent (> 25%). Dominant and rare bacteria did not differ in Bd inhibition in spring peeper and toad populations, in which Bd was absent or at low prevalence (< 7%). In addition, over half of the relative abundance of cultured bacteria on bullfrogs and newts was comprised of inhibitory bacteria, while only 25% and 37% of the relative abundance was inhibitory on spring peepers and toads, respectively. These results suggest that the dominant members of the amphibian skin bacterial community may be functionally important in terms of disease-resistance, and that Bd prevalence and/or host species identity may impact the relative abundance and inhibitory properties of skin bacteria.

Chubs of the genus Nocomis build large pebble nests that may serve as an important microhabitat for macroinvertebrates and small vertebrates in streams of eastern North America. Macroinvertebrates that use chub nests have not been systematically documented. In this study, we examined the macroinvertebrate communities present inside bluehead chub Nocomis leptocephalus nests in a small tributary creek of the New River in Virginia, USA. Individuals from 38 families of macroinvertebrates were recovered from nests. Snails from the family Pleuroceridae were the most numerous invertebrates in four nests examined, making up an average of 13.6 § 7.53% of the macroinvertebrate community. This study revealed that communities in chub nests are diverse but not necessarily unique from the surrounding stream substrate. More longterm studies on macroinvertebrate communities of chub nests are needed to elucidate the function of nests as a stream microhabitat.

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