Environmental DNA (eDNA) methods provide novel options for the detection of pathogens. The amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Ranavirus have been relatively understudied in Texas, US, so we applied eDNA assays for the surveillance of these pathogens in the upper Brazos River basin near the Texas panhandle. We collected water samples from five urban playa lakes and one reservoir in and around Lubbock, Texas. Quantitative PCR detected both Bd and Ranavirus at one playa lake, representing novel detection of both pathogens in the region. Based on these results, we recommend increased monitoring for the pathogens and symptoms of amphibian disease throughout the region.
Texas, US, has been relatively understudied regarding the presence of the amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Ranaviruses. Gaertner et al. (2009) reported infrequent detection of Bd in the Guadalupe and Colorado River basins in central Texas; however, infections were taxonomically widespread, occurring across all five species of narrowly endemic salamanders examined. In a survey of 18 amphibian species in the national forests of east Texas, six species had at least one individual test positive for Bd (Saenz et al. 2010). Ramesh et al. (2013) did not observe Bd infection in amphibians sampled from urban playa lakes in the upper Brazos River basin in the Texas panhandle. No surveys, to our knowledge, have assessed the status of Ranaviruses in Texas. Thus, understanding of amphibian pathogens in Texas and resulting preparedness for future disease outbreaks are limited.
Amphibians in the upper reaches of the Brazos River have been the subject of recent study (Ramesh et al. 2013), but the headwater location of this region makes it a critical site for vigilant monitoring because Bd or Ranavirus emergence in the region could spread across the state through surface water connections. Furthermore, although previous studies have relied on collection and swabbing of individual amphibians for pathogen detection, novel environmental DNA (eDNA) approaches, which collect and analyze genetic material that organisms shed into their local environment (Barnes and Turner 2016), enable an ecosystem-scale approach to surveillance, warranting a revisit of previous study sites to increase understanding of pathogen loads in the environment. Analysis of eDNA has previously successfully been applied to the detection of both Bd (e.g., Kamoroff and Goldberg 2017) and Ranavirus (e.g., Hall et al. 2016), and here, we report on their application in aquatic systems in the upper Brazos River basin, Texas.
We sampled five urban playa lakes in the upper Brazos River basin in and around Lubbock, Texas, which had historically been confirmed to support amphibian populations in multiyear surveys (Ramesh 2012). We also sampled Buffalo Springs Reservoir, which has a surface area of approximately 100 ha and a maximum depth of approximately 16 m (Fig. 1). At each lake, we collected water samples from four sites haphazardly spread out around the shore. At each site, we collected water samples in two different ways. First, for detection of Bd, we collected 250 mL of surface water and conducted on-site filtration using a hand-operated vacuum pump and five 1-µm filters (Nuclepore Track-Etch Membranes, Whatman, Maidstone, UK), with a subsample of 50 mL filtered through each. At the time of collection, the water level at Clapp Park was exceptionally low, resulting in extreme turbidity, precluding efficient filtration. For that reason, only a single 50-mL sample was filtered at each site within Clapp Park. At each lake, we also filtered 250 mL of ultrapure water transported from the laboratory into the field to serve as a negative control (field blank). Filters were transported to the laboratory on ice and stored at –20 C until DNA extraction.
Samples intended for Ranavirus detection were not filtered because we were concerned that even a 1-µm pore would be too large to collect the small virus particles. Instead, for Ranavirus detection, we collected one surface water sample in a 50-mL tube at each site for eDNA extraction via centrifugation and precipitation. Each 50-mL water sample was transported to the laboratory on ice, and a 15-mL subsample was combined with 1.5 mL of 3M sodium acetate and 33 mL of 100% ethanol, following the protocol of Ficetola et al. (2008). Like the filters, these samples were stored at –20 C until DNA extraction.
Finally, at each site, we measured environmental temperature and pH using a handheld probe. We also collected 250 mL of surface water for laboratory quantification of nitrogen (measured as NH3) and phosphorus (measured as PO43–), calcium hardness, and turbidity with a DR3900 spectrophotometer (Hach Company, Loveland, Colorado, USA), a digital titrator (Hach), and a turbidity meter (T100, Oakton Instruments, Vernon Hills, Illinois, USA), respectively.
We extracted eDNA from filters, following the methods of Barnes et al. (2014). Briefly, we began by adding 500 µL of cetyl trimethylammonium bromide cell lysis buffer (CTAB) to each sample, followed by 500 µL of 24:1 chloroform:isoamyl alcohol. Next, we vortexed samples and agitated at a medium speed for 5 min. Samples were then centrifuged at 15,000 × G for 15 min. We transferred 500 µL of the supernatant to a new 1.5-mL tube and added 500 µL of isopropanol and 250 µL of 5M NaCl. Following an overnight incubation at –20 C, we centrifuged samples at 15,000 × G for 15 min to pellet DNA. We decanted the supernatant and washed the pellet twice with 150 µL of 70% ethanol. Finally, we air-dried the DNA pellet and resuspended DNA in 100 µL of low-ethylenediaminetetraacetic acid tris hydrogen chloride (low-TE) buffer. Extracted samples were stored at 4 C.
Water samples collected for eDNA extraction via precipitation were processed similarly to filters, except several additional steps preceded the previously described CTAB-chloroform extraction. First, samples were centrifuged at 4122 × G for 35 min at 6 C to form a pellet. We decanted the supernatant liquid and added 500 µL CTAB cell lysis buffer to each sample, vortexed it, and then transferred the sample to a clean 2-mL tube. The DNA extraction then proceeded identically to the previously described protocol. For both filter and precipitation samples, an empty tube was extracted alongside real field samples and processed through the duration of the experiment as negative controls (extraction blanks).
Detection of Bd (on filters from 250-mL water samples) and Ranavirus eDNA (in material precipitated from 15-mL water samples) was achieved using the quantitative PCR (qPCR) methods described by Kamoroff and Goldberg (2017) and Hall et al. (2016), respectively. Briefly, for each sample, three technical replicate reactions were run on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, California, USA) with the following cycling conditions: 50 C for 2 min, 95 C for 10 min; then, 40 cycles of 95 C for 15 s, and 60 C for 1 min. Each 20-µL reaction included 10 µL of Perfecta Toughmix (Quantabio, Beverly, Massachusetts, USA), 200 nM of each primer and probe, and 4 µL of extracted sample DNA. Triplicate-negative controls (qPCR blanks) featuring low-TE buffer in place of extracted DNA were included on each plate of reactions as well as triplicate positive-control reactions using culture-derived genomic DNA from Bd or Ranavirus. If a sample tested positive for the presence of Bd or Ranavirus, the qPCR was repeated for confirmation. Inhibition in eDNA samples was assessed with TaqMan Exogenous Internal Positive Control Reagents (Applied Biosystems) following manufacturer instructions, and no evidence for inhibition was observed.
Physicochemical conditions were relatively consistent across study sites (Table 1). We did not detect Bd eDNA in any lakes except the lake at Clapp Park (Table 1). Three of the four filtered samples collected at Clapp Park tested positive for the detection of Bd eDNA, and positive detections were repeatable across independent qPCR runs. Similarly, all lakes were negative for the presence of Ranavirus eDNA except Clapp Park (Table 1). Precipitation-extracted samples from all four sites in the lake at Clapp Park tested positive for Ranavirus eDNA, and detections at all four sites were repeatable. All negative controls (i.e., field blanks, extraction blanks, and qPCR blanks) produced negative results, and positive controls amplified as expected.
Detections of Bd and Ranavirus eDNA in Clapp Park, one of many ephemeral playa lakes in the upper Brazos River basin, provide novel evidence for the presence of both pathogens in west Texas and raise concerns about their range and density throughout the state. No survey for the presence of Ranavirus has been done in the region before, to our knowledge, and although Bd has previously been detected in Texas (Gaertner et al. 2009; Saenz et al. 2010), the detection of Bd in our study is particularly surprising because surveys at the same site in 2011–12 failed to detect the pathogen (Ramesh et al. 2013). However, the previous surveillance effort examined individual amphibians for signs of infection, and our study used eDNA methods, which more broadly survey the total environment. Our detections could be the result of increased sensitivity of our method, rather than evidence of novel introduction of the pathogens, so this study justifies revisiting other sites of previous studies and expanding surveillance across the state. Moreover, one caveat of our results is that the presence of Bd and Ranavirus eDNA does not equate to presence of the diseases (Hall et al. 2016), so our study should inspire further monitoring of amphibian populations in the upper Brazos River basin and beyond.
Consideration of Clapp Park sampling conditions provides insight into why this site was the only location in which pathogen eDNA was found. Reservoirs of both Bd and Ranavirus are known to occur in benthic substrates (Johnson and Speare 2005; Gray et al. 2009), and the water level at Clapp Park was notably shallow at the time of sampling; therefore, compared with other sites, samples at Clapp Park were collected closest to the benthos, which unavoidably resulted in a relatively high amount of collected sediments. Increased presence of sediment within the water samples taken from this site (evidenced by extreme turbidity, nitrogen, and phosphorus levels; Table 1) may have contributed to detection of Bd and Ranavirus eDNA. Consequently, future studies should explore whether benthic-focused sampling would increase the potential for detecting these pathogens. Samples across multiple time points for each site will also help determine if pathogens or eDNA detection are influenced by season or other temporal dynamics.
The detection of eDNA of Bd and Ranavirus in Clapp Park also aligns with our understanding of the amphibian community within the site. The American bullfrog (Lithobates catesbeianus), a known carrier of both pathogens (Garner et al. 2006; Lesbarrères et al. 2012), was observed at Clapp Park but not at other sites in the region surveyed by Ramesh (2012). Thus, positive detection of both pathogens at Clapp Park may be explained by the presence of this carrier. This finding further suggests a need for more consideration and conservation of amphibian community compositions in the playa lakes of west Texas and beyond.
This work is a product of the Life Sciences in the 21st century cohort in the undergraduate Program in Inquiry and Investigative Thinking (Pi2) at Texas Tech University and was supported by the Texas Tech University Honors College and Office of the Provost. N. Warren assisted with experimental design. C. Barnes, E. Barnes, and S. Roth assisted with field collections. M. San Francisco provided DNA samples to serve as positive controls in Bd assays, and access to Ranavirus DNA was facilitated by K. Griffis-Kyle and provided by M. Gray and K. Ash. Map of playa lakes of the region depicted in Figure 1 was courtesy of the Playa Lakes Joint Venture (http://pljv.org/). Comments from two anonymous reviewers improved an earlier draft of this manuscript.