Identifying fungal-host associations in an amphibian host system

Abstract

Host-associated microbes can interact with macro-organisms in a number of ways that affect host health. Few studies of host-associated microbiomes, however, focus on fungi. In addition, it is difficult to discern whether a fungal organism found in or on an ectotherm host is associating with it in a durable, symbiotic interaction versus a transient one, and to what extent the habitat and host share microbes. We seek to identify these host-microbe interactions on an amphibian, the Colorado boreal toad (Anaxyrus boreas boreas). We sequenced the ITS1 region of the fungal community on the skin of wild toads (n = 124) from four sites in the Colorado Rocky Mountains, across its physiologically dynamic developmental life stages. We also sampled the common habitats used by boreal toads: water from their natal wetland and aquatic pond sediment. We then examined diversity patterns within different life stages, between host and habitat, and identified fungal taxa that could be putatively host-associated with toads by using an indicator species analysis on toad versus environmental samples. Host and habitat were strikingly similar, with the exception of toad eggs. Post-hatching toad life stages were distinct in their various fungal diversity measures. We identified eight fungal taxa that were significantly associated with eggs, but no other fungal taxa were associated with other toad life stages compared with their environmental habitat. This suggests that although pre- and post-metamorphic toad life stages differ from each other, the habitat and host fungal communities are so similar that identifying obligate host symbionts is difficult with the techniques used here. This approach does, however, leverage sequence data from host and habitat samples to predict which microbial taxa are host-associated versus transient microbes, thereby condensing a large set of sequence data into a smaller list of potential targets for further consideration.

Mr. Toad's wild fungi: Fungal isolate diversity on Colorado boreal toads and their capacity for pathogen inhibition

“…During the summer of 2019, we obtained skin swabs from a subalpine boreal toad site in Chaffee County, Colorado with an approved IACUC protocol (#2629) and an active Colorado Parks and Wildlife scientific collection permit (19HP0998). Since past research has shown the importance of life stage to skin mycobiome composition and diversity (Alexiev et al, 2021;Kueneman et al, 2016b), we sampled at least 3 individuals from each life stage (tadpole, metamorph, subadult, and adult). Individual boreal toads were captured by hand while wearing sterile nitrile gloves and swabbed 10 times using double-headed rayon-tipped swabs (BD BBL, East Rutherford, New Jersey) (n = 16 animals swabbed total).…”

“…We selected representatives from 16 unique fungal isolates, which was a tractable number to work with given laboratory resources. We used previously published ITS marker gene sequences from a past research study of the boreal toad skin mycobiome (sequences are deposited at the European Nucleotide Archive (ENA) in association with the project accession PRJEB41738), which identified fungal OTU's that were associated with the host (at each distinct life stage) as opposed to its habitat (Alexiev et al, 2021). Although we cultured and sequenced 120 distinct isolates, we did not have the capacity to test each against Bd at the time, so we used our previous study results to choose a tractable number to test.…”

“…Although we cultured and sequenced 120 distinct isolates, we did not have the capacity to test each against Bd at the time, so we used our previous study results to choose a tractable number to test. Therefore, we then chose cultured isolates to test against Bd in co-culture in the present study based on groups that were observed to be host-associated or consistently present across most boreal toad life stages in the previous study (Alexiev et al, 2021). Once we had Sanger sequences of the fungal isolates, we created a custom BLAST database of the Sanger sequences from fungal isolates in this study (amplified ITS1F-ITS4 with average read length of 583 base pairs), which are longer than the high-throughput sequences (amplified ITS1F-ITS2 with average read length of 158 base pairs), and matched the ITS high-throughput sequence data from the 2021 study to these (a histogram of percent identity matches is shown in Suppl Fig.…”

“…For each sequence, we identified the host's stage of development (i.e., eggs, tadpoles, metamorphs, etc.) and how the sequence identities compared to ITS high-throughput sequence whole community data from Alexiev et al, 2021. We compared our findings to data demonstrating whether ITS-identified OTUs were host-or habitat-associated (Alexiev et al, 2021) and then plotted how many of these were Sanger-sequenced and had consistent phylogenetic identification.…”

Together or Alone: Evaluating the Pathogen Inhibition Potential of Bacterial Cocktails against an Amphibian Pathogen

“…Thus, our work investigates how the J. lividum strain BTP, which is specifically found on this host, and other skin-associated bacteria interact with Bd on the Colorado boreal toad ( Anaxyrus boreas boreas ), which is a high-elevation wetland amphibian that is locally endangered in the Rocky Mountains due to Bd ( 41 – 44 ). Research on this species to date has prioritized the use of next-generation sequencing and in vivo treatments to develop an effective probiotic bioaugmentation strategy for boreal toads ( 12 , 34 , 35 , 45 – 47 ). Adapting probiotic treatments to field conditions in wild populations remains as ongoing work.…”