1. Freshwater Mussel Collaborative Study for Wastewater Treatment Plants May 23, 2016 Validation of Environmental DNA for Determining Presence of Unionid Mussels in a River Environment

2. Background and Purpose U.S. EPA’s 2013 ammonia criteria are based on presence/absence of unionid freshwater mussels U.S. EPA’s 2013 ammonia criteria are based on presence/absence of unionid freshwater mussels Central Valley RWQCB requires determination of mussel presence/absence in receiving waters for wastewater treatment facilities Central Valley RWQCB requires determination of mussel presence/absence in receiving waters for wastewater treatment facilities CVCWA and 42 Central Valley wastewater agencies formed a collaborative effort to identify the best methods for determining mussel presence/absence CVCWA and 42 Central Valley wastewater agencies formed a collaborative effort to identify the best methods for determining mussel presence/absence Environmental DNA (eDNA) identified as a cost-effective method for defensible presence/absence determinations Environmental DNA (eDNA) identified as a cost-effective method for defensible presence/absence determinations eDNA Pilot Study – evaluate the efficacy of using eDNA in Central Valley receiving waters eDNA Pilot Study – evaluate the efficacy of using eDNA in Central Valley receiving waters

3. Environmental DNA Aquatic organisms emit DNA into water column Aquatic organisms emit DNA into water column Easily sampled = low field costs Easily sampled = low field costs Low analytical costs (qPCR) Low analytical costs (qPCR) No direct observation or handling of specimens No direct observation or handling of specimens Ideal for rare or cryptic organisms Ideal for rare or cryptic organisms Detectable on a species-specific basis: Detectable on a species-specific basis: Presence / absence Presence / absence DNA load (“signal strength”) DNA load (“signal strength”)

4. eDNA Pilot Study Initial Task: Methodology Setup for eDNA Analyses Initial Task: Methodology Setup for eDNA Analyses Four Study Elements: Four Study Elements: 1. Pit River Sampling for eDNA Method Validation 2. WWTP Receiving Water Sampling 3. Delta Sampling 4. Evaluation of Mussel eDNA Attenuation

5. Methodology Setup for eDNA Analyses Mussel Assay Development AssayCladeSpecies Detected Occurrence in the Central Valley Anodonta Clade 1 Anodonta californiensis Yes Clade 1 A. nuttalliana Yes Clade 1 A. wahlamatensis Yes * Clade 2 A. oregonensis Yes n/a A. impura No Gonidean/a Gonidea angulata Yes Margaritiferan/a Margaritifera falcata Yes * Species is now considered genetically indistinct from A. nuttalliana.

6. Methodology Setup for eDNA Analyses Assay Validation All three genera present in the Pit River, CA All three genera present in the Pit River, CA Collected tissue samples from 3 specimens per genus (9 tissue samples total) Collected tissue samples from 3 specimens per genus (9 tissue samples total) DNA extracted from each of the nine mussel tissue samples tested with each assay DNA extracted from each of the nine mussel tissue samples tested with each assay All nine samples tested positive for its respective assay and negative for the others All nine samples tested positive for its respective assay and negative for the others Negative control assays (blank water): no false-positives (non-specific bindings) occurred in the qPCR process Negative control assays (blank water): no false-positives (non-specific bindings) occurred in the qPCR process Conclusion: eDNA assays were performing properly Conclusion: eDNA assays were performing properly

7. Study Element 1: Pit River Sampling Approach Review available Pit River mussel records Review available Pit River mussel records Identify four candidate zones Identify four candidate zones Select two suitable study zones Select two suitable study zones All three mussel genera present All three mussel genera present Accessible Accessible Confirm presence of all three genera via snorkel surveys at one transect per zone Confirm presence of all three genera via snorkel surveys at one transect per zone Sample three transects per zone Sample three transects per zone

8. Study Element 1: Pit River Sampling Methods ZoneTransect Snorkel Survey to Confirm Presence of All Mussel Genera? 1 1Yes 2No 3 3 4Yes 5No 6 At each transect, three 1-L samples and one 4-L sample was collected (7 L total) At each transect, three 1-L samples and one 4-L sample was collected (7 L total) One 1-L field blank was collected using distilled water at each zone (false-positive assessment) One 1-L field blank was collected using distilled water at each zone (false-positive assessment)

9. Study Element 1: Pit River Sampling Sample Locations – October 2015 Zone 1 Zone 3

10. Study Element 1: Pit River Sampling Pit River Zone 1 eDNA Results Transect eDNA Load (Log(Sequences/Liter)) AnodontaGonideaMargaritifera 1 Surveyed 9.69.810.3 10.010.210.4 10.1 10.6 10.210.410.7 2 Not Surveyed 10.19.910.8 10.29.910.9 9.78.710.9 10.09.710.8 3 Not Surveyed 9.69.79.8 9.19.510.9 9.28.910.1 9.69.410.7

11. Study Element 1: Pit River Sampling Pit River Zone 3 eDNA Results Transect eDNA Load (Log(Sequences/Liter)) AnodontaGonideaMargaritifera 4 Surveyed 9.710.68.8 8.96.48.7 9.610.79.6 9.79.88.9 5 Not Surveyed 4.74.54.8 9.410.59.2 9.310.2ND 9.410.19.1 6 Not Surveyed ND

12. Study Element 1: Pit River Sampling Signal Strength Overall average eDNA concentration by genus (log(DNA sequences/L): Overall average eDNA concentration by genus (log(DNA sequences/L): Margaritifera: 10.6 Margaritifera: 10.6 Gonidea: 10.1 Gonidea: 10.1 Anodonta: 9.7 Anodonta: 9.7 Transect 1 (Malinda Gulch) Spring Rivers Ecological Services 2015 mussel survey: Transect 1 (Malinda Gulch) Spring Rivers Ecological Services 2015 mussel survey: Margaritifera: n = 1,800 Margaritifera: n = 1,800 Gonidea: n = 865 Gonidea: n = 865 Anodonta: n = 13 Anodonta: n = 13

13. Study Element 1: Pit River Sampling Transect 1 Load-Abundance Relationship

14. Summary DNA from tissue samples collected from Pit River Anodonta, Gonidea, and Margaritifera was successfully amplified by the respective qPCR assay DNA from tissue samples collected from Pit River Anodonta, Gonidea, and Margaritifera was successfully amplified by the respective qPCR assay eDNA successfully detected mussels at all sites where presence was confirmed visually (i.e., no false- negatives) eDNA successfully detected mussels at all sites where presence was confirmed visually (i.e., no false- negatives) Analyses of field blanks yielded no false-positives Analyses of field blanks yielded no false-positives No eDNA detected in non-flowing habitats (Transect 6) No eDNA detected in non-flowing habitats (Transect 6) eDNA concentrations were generally commensurate with abundance of each mussel genus eDNA concentrations were generally commensurate with abundance of each mussel genus