Supporting Information

Transcription

1 Supporting Information Environmental DNA (edna) Shedding and Decay Rates to Model Freshwater Mussel edna Transport in a River Brandon J. Sansom 1 and Lauren M. Sassoubre 1* 1 Department of Civil, Structural, and Environmental Engineering, The State University of New York at Buffalo, Buffalo, New York * Corresponding author. Tel: Fax lsassoub@buffalo.edu Supporting Information contains: Number of pages: 8 Number of tables: 2 Number of figures: 3 S1

2 Materials and Methods Genus-specific assay design and QPCR optimization for Lampsilis siliquoidea To develop species specific primers, a representative mitochondrial sequence for the L. siliquoidea was downloaded from the National Center Biotechnology Information (NCBI) Genbank. 1 We specifically targeted the NADH gene and the cytochrome c oxidase subunit 1 (COI) gene because of sequence availability in Genbank. Primers and an internal hybridization oligo or probe were designed using PrimerBlast 2 with default parameters except: (1) primer melting temperature range C, (2) probe melting temperature range C, (3) GC content 35%-65% for both primers and probe, and (4) primer pair specificity checking against other mussel genera found in the same environment. The primer probe sets returned by the program were evaluated for the following criteria: (1) GC content around 50%, (2) a melting temperature between the primers less than 5 C, (3) a melting temperature between the primers and probe of 8-10 C, (4) primer and probe length, (4) no runs of 4 or more of the same base, (5) avoid a T base at the 3 end of the primers, (6) a C or G base at the 3 end of the primers, (7) no more than 3 C and/or G bases in the last 5 base pairs at the 3 end of the primers, and (8) avoid a G base at the 5 end of the probe (to avoid quenching of the fluorophore). Primer probe sets matching all the criteria were mapped onto an alignment made using Clustal Omega 3-5 of sequences from both target and non-target species to look for mismatches in the primer and probe regions. Primer and probe sets that were specific to the target species and contained the most mismatches with other non-target species were ordered from Integrated DNA Technologies (IDT) and tested with genomic DNA in the lab. Data Analysis The error associated with the shedding rate was determined by propagating errors associated with k, C, and V: δs S = ( δk k ) 2 + ( δc C ) 2 + ( δv V ) 2 (eqn. S1) where δ represents the standard deviation associated with each variable. Mass Balance Model and Derivation A simplified, one-dimensional plug-flow reactor model (equation 3) was developed to model L. siliquoidea edna as a function of distance from a mussel bed. The mass balance equation is a modification of the Navier-Stokes advection-diffusion equation for the x direction only: C C + u t x = E 2 C x x 2 kc (eqn. S2) where C represents the edna concentration (copies per ml), t represents time in days, u is the water velocity (m/s) in the streamwise direction (x-direction), E x is the diffusion coefficient in the streamwise direction, and k is the first-order decay constant. We assumed that the diffusive forces were negligible compared to advective forces, and thus simplified the equation to: C C + u = kc (eqn. S3) t x S2

3 Further, we assumed that the edna being released in the stream we are modeling is at steady state ( dc dt = 0): u C x = kc (eqn. S4) The steady state solution to equation S4 is: C = C o e kx u (eqn. S5) Here, C o represents the initial L. siliquoidea edna concentration in our stream. The concentration of L. siliquoidea edna in the environment depends on six main factors: 1) the population size, 2) the shedding rate, 3), the decay rate, 4), the advective forces of flow, 5) the distance edna is collected from its source, and 6) the time between when DNA is shed and when it is collected. The decay rate (k), advective force (u), and distance (x) are factors directly represented in equation S5. To initialize our model, we first had to make certain assumptions about C o to characterize the mussel bed, including population size, shedding rate, and time. To better represent this initial concentration at the mussel bed, we let C o = C bed and wrote C bed as a function of shedding (S, copies/hour/mussel), population size (M, mussels/m 3, and time (t, hours): C bed = S M t (eqn. S6) Because we did not observe statistical differences in shedding rates between mussel densities, we can assume that each mussel sheds a similar amount of edna on a per mussel basis. Therefore, we averaged the steady state concentration of edna (copies per hour per mussel) from the shedding and decay experiments performed in this study and input this value as our shedding rate (S ) in equation S6. In traditional mussel surveys, the population size of mussels is represented in units of mussels/m 2. While this is a good index to consider the size of a given population for sedentary organisms that live in the substratum, edna fragments are shed into and quickly mixed in the water column, especially in turbulent flows of natural streams. Therefore, to better represent how much edna is being shed into a volume of water, we represent the population size as mussels/m 3. From a practitioner standpoint, achieving units of mussels/m 3 is as simple as dividing the density of mussels in units of mussels/m 2 by the mean water depth (m). Finally, we observed a sharp increase in edna concentration initially after the mussels were added to the experimental tanks. In all experiments, the concentration leveled off after 12 hours and remained steady for the duration the mussels were in the mesocosms. Therefore, we assumed that steady state is reached within 12 hours, and used a time of 12 hours in our model. S3

4 Results and Discussion Figure S1: The qpcr calibration curve using gblock Gene Fragments synthesized by IDT (IDT, Coralville, IA) sequences. The pooled standard curves had a slope = and intercept = 33.1, resulting in an assay efficiency of 99.0% (r 2 = 0.93). Figure S2: edna concentration from the field samples collected from Tonawanda Creek as a function of distance downstream of a mussel bed. Error bars represent ± standard error of three biological samples, each ran in triplicate qpcr measurements. The predicted edna concentration determined by a onedimensional model is represented by the solid line. S4

5 Figure S3: The predicted edna concentration as a function of distance downstream of a mussel bed. Vertical dashed lines indicate the predicted downstream detection distance for varying volumes of water filtered. S5

6 Table S1. Sample collection information for shedding and decay experiments conducted in 2016, including date and time, hours since the start of the experiment, and presence or absence of Lampsilis siliquoidea in the tank. The bolded time points indicate when the edna concentration in the tank appeared to be at steady state where shedding balanced decay. Sample Number Moderate Density (replicate 1: 55 /m 2 experiment conducted from 09/17/ /18/2016) Start (hours) High Density (110 /m 2 experiment conducted from 09/17/ /18/2016) Start (hours) Environmental Treatment (Tonawanda Creek Water experiment conducted from 09/19/ /18/2016) Start (hours) 0 0:00 No 0:00 No 0:00 No 1 14:00 Yes 14:00 Yes 5:00 Yes 2 21:00 Yes 21:00 Yes 18:00 Yes 3 28:00 Yes 28:00 Yes 24:00 Yes 4 38:15 Yes 38:15 Yes 31:00 Yes 5 45:00 Yes 45:00 Yes 41:00 No 6 50:00 Yes 50:00 Yes 47:30 No 7 63:00 Yes 63:00 Yes 52:30 No 8 69:00 Yes 69:00 Yes 65:00 No 9 76:00 Yes 76:00 Yes 71:00 No 10 86:00 No 86:00 No 78:00 No 11 92:30 No 92:30 No 89:30 No 12 97:30 No 97:30 No 95:30 No :00 No 110:00 No 99:00 No :00 No 116:00 No 114:15 No :00 No 123:00 No 124:15 No :30 No 134:30 No 137:45 No :30 No 140:30 No 149:00 No :00 No 144:00 No 162:15 No :15 No 159:15 No 170:00 No :15 No 169:15 No 336:30 No :45 No 182:45 No 501:00 No :00 No 194:00 No 697:00 No :15 No 207:15 No :00 No 215:00 No :30 No 381:30 No :00 No 546:00 No :00 No 742:00 No S6

7 Table S2. Sample collection information for shedding and decay experiments conducted in 2017, including date and time, hours since the start of the experiment, and presence or absence of Lampsilis siliquoidea in the tank. The bolded time points indicate when the edna concentration in the tank appeared to be at steady state where shedding balanced decay. Sample Number Low Density (replicate 1: 16 /m 2 experiment conducted from 08/23/2017-9/21/2017) Start (hours) Low Density (replicate 2: 16 /m 2 experiment conducted from 08/23/2017-9/21/2017) Start (hours) Moderate Density (replicate 2: 55 /m 2 experiment conducted from 08/23/2017-9/21/2017 Start (hours) 0 0:00 No 0:00 No 0:00 No 1 16:25 Yes 16:25 Yes 16:25 Yes 2 23:55 Yes 23:55 Yes 23:55 Yes 3 40:25 Yes 40:25 Yes 40:25 Yes 4 48:25 Yes 48:25 Yes 48:25 Yes 5 64:25 Yes 64:25 Yes 64:25 Yes 6 74:10 Yes 74:10 Yes 74:10 Yes 7 87:55 No 87:55 No 87:55 No 8 98:05 No 98:05 No 98:05 No 9 112:25 No 112:25 No 112:25 No :55 No 120:55 No 120:55 No :40 No 136:40 No 136:40 No :25 No 145:25 No 145:25 No :55 No 160:55 No 160:55 No :25 No 168:25 No 168:25 No :25 No 183:25 No 183:25 No :40 No 361:40 No 361:40 No :25 No 695:25 No 695:25 No S7

8 References 1. Benson, D. A.; Karsch-Mizrachi, I.; Lipman, D. J.; Ostell, J.; Wheeler, D. L., GenBank. Nucleic acids research 2005, 33, (D1), D Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T. L., Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC bioinformatics 2012, 13, 134. S8