Table 2.

Validation of water samples from the Zebrafish International Resource Center (ZIRC) using eDNA assay for Pseudoloma neurophilia. For every filter, triplicate PCR reactions were executed; 2 of the 3 reactions must result in a positive detection to be deemed positive. Negative control samples include ZIRC samples (con O and con P), which are post-filtration water from two recirculating systems: System A and B. Con T = laboratory water. Dpf = days post fertilization. * Indicates that the filter was deemed positive for detection of the parasite. Estimates of the prevalence of infection was determined based on ZIRC histology or tissue PCR results. Letters (column 1) are the designations for tanks that were sampled. PCR values are reported as copy number/L.

Tank	ZIRC Stock (System)	Age (dpf)	No. of Fish in Tanks	ZIRC Diagnostic Prevalence	WB-qPCR Prevalence	Filter	dPCR result (copy #/L) Replicate 1	dPCR result (copy #/L) Replicate 2	dPCRresult (copy #/L) Replicate 3
M	ZS 11236 (A)	105	316	N/A	2/10	1	0	0	0
						2*	0.86	0.86	0
						3	0	0	0.86
						4	0	0	0
N	ZS 11015 (B)	251	279	1/5 histo	8/10	1*	12.92	20	12.08
						2*	6.88	12.06	7.76
						3*	7.76	2.58	7.76
						4*	11.46	7.74	6.02
Q	ZS 11229 (A)	253	255	6/11 histo	8/10	1*	5.16	6.04	3.46
						2*	1.72	1.72	0.86
						3*	1.72	2.58	1.72
						4*	0.88	0	0.86
R	ZS 11229 (A)	253	253	1/11 histo	6/10	1*	0.86	2.58	3.94
						2*	2.58	0	2.58
						3*	6.02	7.76	3.44
						4*	0	0.86	0.86
S	ZS 11229 (A)	253	291	7/11 histo	9/10	1*	3.44	0.86	0
						2*	0	0.86	0.86
						3	0	0	0.86
						4*	0.88	4.3	0.86
U	ZS 11403 (A)	128	235	0/11 PCR	–	1	0	0	0
						2	0	0	0
V	ZS 11403 (A)	128	259	0/11 PCR	–	1	0	0	0
						2	0	0	0
W	ZS 11403 (A)	128	287	0/11 PCR	–	1	0	0	0
						2	0	0	0
X	ZS 11403 (A)	128	285	0/11 PCR	–	1	0	0.86	0
						2	0	0.86	0
Y	ZS 11522 (A)	266	260	9/11 histo	10/10	1*	12.06	2.6	8.66
						2*	8.62	3.44	6.9
Z	ZS 11522 (A)	266	170	7/11 histo	7/10	1*	19.82	24	18.1
						2*	15.86	28	24
AA	ZS 11497 (B)	281	229	4/5 histo	6/10	1*	0.86	0	0.86
						2	0	0	0
CONO	System B post filtration	–	–	–	–	1	0	0.086	0
						2	0	0	0
CONP	System A post filtration	–	–	–	–	1	0	0	0
						2	0	0	0
CONT	System A post filtration	–	–	–	–	1	0	0	0
						2	0	0	0