How should we sample snail microbiomes?

Background: The microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the bacterial 16S V4 ribosomal DNA of the hemolymph (blood) of Biomphalaria spp. snails, one of the vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs. Results: We dissected ten snails for each of two different species (B. alexandrina and B. glabrata) and collected their organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S V4 rDNA revealed organ-specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome. Conclusions: These results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

Leveraging preserved specimens of Nerodia to infer the spatiotemporal dynamics of Ophidiomyces ophidiicola via quantitative polymerase chain reaction.

Ophidiomyces ophidiicola (Oo) is a fungal pathogen and the causative agent of ophidiomycosis that has affected multiple snake taxa across the United States, Europe, and Asia. Ophidiomycosis has often been referred to as an emerging infectious disease (EID); however, its status as an EID has recently come under debate. Oo infections have been confirmed in wild snake populations in Texas; however, it is unknown if the pathogen is novel (i.e., invasive) or endemic to the state. To address this knowledge gap, we conducted surveys for Oo among preserved Nerodia deposited at three university museums in Texas. First, we visually assessed snakes for signs of infection (SOI), and if SOI were present, we sampled the affected area. We then used quantitative polymerase chain reaction to diagnose the presence of Oo DNA on areas with SOI and used these data to evaluate spatiotemporal patterns of Oo prevalence. We also tested for significant spatial clusters of Oo infenction using a Bernoulli probability model as implemented in the program SatScan. We found that the proportion of snakes exhibiting SOI was constant over time while the prevalence of Oo DNA among those SOI increased across space and time. Within these data, we detected an incidence pattern consistent with an introduction and then spread. We detected six spatial clusters of Oo infection, although only one was significant. Our results support the hypothesis that Oo is an emerging, novel pathogen to Texas snakes. These data narrow the knowledge gap regarding the history of Oo infections in Texas and establish a historical record of confirmed Oo detections in several counties across the state. Thus, our results will guide future research to those areas with evidence of past Oo infections but lacking confirmation in contemporary hosts.