Diet diversity and environment determine the intestinal microbiome and bacterial pathogen load of fire salamanders

Abstract

Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

COI Metabarcoding Provides Insights into the Highly Diverse Diet of a Generalist Salamander, Salamandra salamandra (Caudata: Salamandridae)

“…This seems a probable explanation for the observed prey richness among samples from Ons sampled during autumn. Relatively few arachnid (Arachnida) and centipede (Chilopoda) taxa were identified among our samples, although they have been reported in the diets of salamanders [15,22], suggesting an absence either among our samples or in local abundance.…”

“…However, in many of these cases, often only a few prey taxa could be identified, because of either digestion or low taxonomic resolution. Our study identified 58 different prey taxa from three populations at variable taxonomic resolutions-fewer than the 76 prey taxa identified by gastric inspection from a higher number of individuals (N = 72), localities (N = 10), and a wider environmental and ecological survey [15], but nearly threefold more than from previous taxonomic assignment using DNA metabarcoding of 18S across three forest populations in Belgium [22]. Thus, DNA metabarcoding of COI increases taxonomic resolution and provides a cost-effective and expedient method for characterizing the diets of salamanders.…”

“…We observed this anecdotally in the prevalence of Coleoptera identified by Bas et al [15], of which 16 genera were identified, as compared to the four genera identified in this study, with both studies identifying Nalassus as the most common prey among the class Insecta. Conversely, 18S metabarcoding by Wang et al [22] found that the most common prey were Gastropoda, which far exceeded other prey in prevalence. Gastropoda have been reported as a common prey elsewhere [16,50], as well as in our results.…”

“…OTUs were assigned to a taxon using BOLDIGGER v.1.2.5 [43]. OTUs with a minimum of 90% similarity to a taxon included in the phyla Annelida, Arthropoda, or Mollusca were retained as plausible invertebrate prey [22]. Samples with less than 100 reads assigned to dietary OTUs were discarded, as were OTUs comprising less than 1% of the total dietary reads per sample, so as to avoid errors from tag jumping or overrepresentation of rare prey [44].…”

“…To our knowledge, only one study has applied DNA metabarcoding to investigate the diet of adult salamander species. Specifically, Wang et al [22] used the 18S ribosomal RNA (18S) region to characterize the diets of adult fire salamanders (Salamandra salamandra) (Linnaeus, 1758) by collecting fecal samples from three Belgian forests. While 18S has proven useful to detect potential prey items, the use of more informative (i.e., variable) DNA fragments such as the cytochrome c oxidase I (COI) benefits from a large reference database that is supported by the Barcode of Life Data System (BOLD) [23,24], and the relatively high variability of the region allows for high-resolution taxonomic assignment [5,[25][26][27].…”