Sensitive detection of rare species is challenging in aquatic environments, but essential for informed conservation of endangered species and proactive management responses for invasive species that threaten native communities. Environmental DNA (eDNA) is a recent technique being used to detect rare aquatic species and determine habitat occupancy from water samples (Ficetola et al. 2008; Goldberg et al. 2011; Jerde et al. 2011). We made use of the growing global DNA barcoding database (BOLD; Ratnasingham and Hebert 2007) to design 36 species-specific primer pairs for eDNA detection of 4 endangered freshwater species and 10 aquatic invasive species that are considered threats to Laurentian Great Lakes ecosystems (Table 1).

Table 1 Species, accession number, primer sequence, fragment size, annealing and melting temperature (TA and TM), % GC content and cross-species amplification results for 36 COI primer pairs developed to detect rare aquatic species from environmental DNA. “Cross species” indicates non-target species tested for amplification; “–” no amplification; “n/a” no species tested
Full size table

Species-specific primers were designed to target small fragments (80–280 bp) within the 5′ barcoding segment of the COI gene (Hubert et al. 2008). Primer-BLAST (Ye et al. 2012; http://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to identify binding sites and verify specificity in silico against available sequence data (GenBank http://www.ncbi.nlm.nih.gov/genbank and BOLD http://www.boldsystems.org). Mitochondrial sequence data from GenBank and BOLD were used for target sequences (Table 1). User-defined primer parameters included product size range of 80–280 bp, 10 candidate primer pairs, and a minimum of 3 mismatches with unintended targets. Primer pair specificity checking was enabled against all sequence data in the nr database (http://www.ncbi.nlm.nih.gov/genbank). Targets with 8 or more mismatches were ignored. Primer-BLAST default parameters were used for all remaining parameters.

Primer pairs were selected for testing based on target position, fragment size, low self complementarity, and no unintended targets in the sampling region. Degenerate bases were incorporated at positions where intraspecific polymorphisms were identified in priming sites (primers: DbuCOI3F, DpoCOI10R, HanCOI8R, and HanCOI9F/R). Forward primers were ordered with an M13 sequence tail for future use on automated sequencers with the exception of 11 primers; HnoCOI1, HnoCOI2, HnoCOI5, HmoCOI1, HmoCOI2, HmoCOI5, CarCOI2, CarCOI3, CidCOI1, CidCOI6, CidCOI7.

Genomic DNA was extracted from preserved tissues following an alkali extraction protocol (Malago et al. 2002) and stored at −20 °C until primer testing. Each primer set was screened for amplification using tissue-extracted DNA from 3 to 4 individuals. Preliminary primer screening for amplification, annealing temperature, and specificity (when applicable) was performed in 10 μL PCR reactions. Reaction cocktails contained template DNA, 2 μL 5× PCR Buffer (Promega), 1.5 mM MgCl2 (Promega), 0.2 mM of each dNTP, 0.2 mg/mL BSA (BioShop), 0.2 μM of each primer (Eurofins MWG Operon), 0.25 U of Taq polymerase (Promega), and ddH2O to a final reaction volume of 10 μL. Thermocycler conditions for PCR were 94 °C for 180 s followed by 35 cycles of 94 °C for 45 s; 48–70 °C for 45 s, and 72 °C for 60 s, followed by a final extension at 72 °C for 10 min. Amplicons stained with SyBr Green (Cedarlane Laboratories) were visualized by electrophoresis on a 1.5 % agarose gel and size referenced against a 100 bp size standard (BioShop). Positive amplification was recognized as a single band at the expected fragment size (Table 1). Optimal annealing temperature was determined by high DNA yield and amplicon quality. Negative controls were included in all reactions and run on each gel. Select primer pairs were validated for species specificity using tissue derived DNA from closely related and/or co-occurring species as well as the target species (Table 1).

All primer pairs successfully amplified DNA of the expected fragment length from tissue derived DNA from the target organism. Twenty-six primer pairs were selected to be tested for cross-species amplification with DNA (or eDNA) from closely related or co-occurring species. Eighteen of the 26 primer pairs were species specific (Table 1). Non-target species either failed to amplify or amplified only at lower annealing temperatures, with exclusion of non-target amplification at higher annealing temperatures (Table 1). Eight primer pairs consistently amplified non-target species at the expected fragment length at all annealing temperatures. Surprisingly, four of these primers cross amplified non-target species from a different genus (Table 1); the remaining four primer pairs amplified non-target species from within the same genus. Non-specificity may be advantageous when screening for multiple species. For instance, both silver and bighead carp DNA amplified with three primer pairs (two designed to target silver carp and one designed to target bighead carp). The objective of current Great Lakes surveillance efforts is to detect any of three invasive Asian carp species (www.asiancarp.us). Therefore primary amplification with a genus-specific primer set may be more efficient for a widespread screening process, followed by standard sequencing of resulting positives for species verification. Eight primer pairs targeting a total of three species were not tested for cross-species amplification because target species were only distantly related to potential co-occurring species as identified in Hubert et al. (2008).

The development of species-specific primers to detect aquatic species of concern should greatly assist management to address challenges facing Great Lakes biodiversity. In general, species-specific primers targeting the mitochondrial genome are valuable tools for the emerging use of eDNA for detection of rare aquatic species (Ficetola et al. 2008; Darling and Mahon 2011). The global barcoding effort provides the much needed groundwork to efficiently incorporate eDNA analysis into biodiversity monitoring and surveillance efforts.