Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability

Abstract

Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen Batrachochytrium dendrobatidis. Yet there has been no systematic survey of anti-B. dendrobatidis bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to B. dendrobatidis. Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low B. dendrobatidis prevalence. We determined the numbers and identities of anti-B. dendrobatidis bacteria on 61 Plethodon salamanders (37 P. cinereus, 15 P. glutinosus, 9 P. cylindraceus) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled P. cinereus along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti-B. dendrobatidis bacterial operational taxonomic units (OTUs) and found that the degree of B. dendrobatidis inhibition was not correlated with relatedness. Five anti-B. dendrobatidis bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti-B. dendrobatidis bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti-B. dendrobatidis bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti-B. dendrobatidis bacteria per individual ( ϭ 3.3) than those at Catoctin MP ( ϭ 0.8) and at Mt. Rogers NRA ( ϭ 0.4). All salamanders tested negative for B. dendrobatidis. Anti-B. dendrobatidis bacterial species are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a protective role for these salamanders.IMPORTANCE Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen, Batrachochytrium dendrobatidis. Some amphibians die from B. dendrobatidis infection, whereas others do not. The bacteria that can kill B. dendrobatidis, called anti-B. dendrobatidis bacteria, are thought to influence the B. dendrobatidis infection outcome for the amphibian. Yet how anti-B. dendrobatidis bacterial species vary among amphibian species and populations is unknown. We determined the distribution of anti-B. dendrobatidis bacterial species among three salamander species (n ϭ 61) sampled at three localities. We identified 50 unique anti-B. dendrobatidis bacterial species and found that all of the tested salamanders were negative for B. dendrobatidis. Five anti-B. dendrobatidis bacterial species were commonly detected, suggesting a stable, functional association with these salamanders. The number of

Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

“…We quantified the overall summed abundance of putatively anti‐Bd bacterial OTUs, and refer to this as abundance of putative anti‐Bd OTUs. To identify putative anti‐Bd OTUs, we followed methods as outlined by Muletz‐Wolz, DiRenzo, et al () to identify bacterial OTUs that were taxonomically similar to a culturable anti‐Bd bacterial database (Woodhams et al, ), which included sequences of anti‐Bd bacteria we have isolated from Plethodon salamanders (Muletz‐Wolz, DiRenzo, et al, ). We used two levels to match our bacterial OTUs to the culturable anti‐Bd bacteria database: matching at ≥97% sequence similarity and matching at ≥99% sequence similarity (Table ).…”

“…For instance, red‐backed salamanders ( P. cinereus ) are putatively resistant to Bd; they are rarely infected with Bd in the wild even though Bd occurs throughout their range (Muletz, Caruso, Fleischer, McDiarmid, & Lips, ) and they can clear moderate infection loads in lab experiments (Becker, Brucker, Schwantes, Harris, & Minbiole, ; Becker & Harris, ; Muletz et al, ). The skin microbiome of P. cinereus may mediate the effects of Bd infection by having a high abundance of bacteria that can kill Bd (referred to as anti‐Bd bacteria; Becker & Harris, ; Harris, Lauer, Simon, Banning, & Alford, ; Muletz‐Wolz, DiRenzo, et al, ; Muletz‐Wolz, Yarwood, Campbell Grant, Fleischer, & Lips, ). Bd loads on wild‐caught P. cinereus and other species co‐occurring in their range are usually low (Muletz et al, ; Muletz‐Wolz, Yarwood, et al, ; Richards‐Hrdlicka, Richardson, & Mohabir, ), suggesting an enzootic stage of Bd in the eastern USA.…”

Low-load pathogen spillover predicts shifts in skin microbiome and survival of a terrestrial-breeding amphibian

“…We focused on frog assemblages from Brazil's Atlantic forest, a biodiversity hotspot highly impacted by Bd [17], and took into account that the cutaneous microbiomes of amphibians can play a role in their defenses against the disease chytridiomycosis [20][21][22]. We selected four aquatic-breeding amphibian species with varying life histories (stream and pond breeders; tree frogs and frogs), and one focal terrestrial-breeding species: the pumpkin toadlet Brachycephalus pitanga.…”

Land cover and forest connectivity alter the interactions among host, pathogen and skin microbiome

“…Because amphibian skin microbes and Bd may occupy the same microenvironment (epidermal tissue), the amphibian skin microbiome could play a vital role in host defences against Bd [21,22]. Recent studies have reported strong inhibitory effects of particular bacterial species on Bd, both in culture [23][24][25] and in vivo [6,7]. Bacterial diversity was also linked to Bd suppression in experimentally assembled biofilm communities [26].…”