title: Placobdella parasitica
design: Leech specimens were collected from the underside of rocks, wood and other debris in Silvermine Lake, Harriman State Park, NY, USA (Placobdella ali), Lake Skannatati, Harriman State Park, NY, USA (Placobdella parasitica) and Squires Lake, Whatcom County, WA, USA (Placobdella kwetlumye), and immediately preserved in RNAlater (Life Technologies / Thermo Fisher Scientific). Specimens were identified using a stereomicroscope while submerged in RNAlater; identifications were later verified molecularly using mitochondrial cytochrome c oxidase subunit I (COI) and nuclear NADH dehydrogenase 1 (ND1). Prior to RNA extraction, leeches were washed in 0.5% bleach for 1 min and subsequently rinsed in deionized water for 1 min to minimize contamination with surface bacteria. Using sterilized dissecting tools, salivary tissue masses (glandular tissue) from single individuals of each species were removed aseptically and subsequently rinsed in 0.5% bleach for 1 min followed by rinsing in deionized water for 1 min. Total RNA then was isolated using a hybrid protocol as described in Kvist et al. (2014). Total RNA was visualized on an Agilent 2100 BioAnalyzer (Agilent Technologies) using an Agilent RNA 6000 Nano Kit. First and second strand cDNA synthesis followed the protocol described in Kvist et al. (2014), which is a modified protocol originally described by Hale et al. (2009). Second strand cDNA was quantified on a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific) and visualized using a 1.1% agarose gel in 1XTBE buffer and an Agilent High Sensitivity DNA Kit.    Library construction, multiplexing, emPCR, and pyrosequencing  Approximately 500 ng (in 100 μL) of each cDNA library (n=3) was processed individually following the manufacturer's protocols as outlined in the Roche 454 RL Preparation Method Manual (Roche Applied Sciences; version June 2010). Following nebulization and fragment end repair, RL MID (barcoded) adaptors were ligated to each library. Prior to pooling, the individually barcoded libraries underwent small fragment removal and library assessment/quantitation using an Agilent 2100 Bioanalyzer and QuantiFluor ST Fluorometer (Promega); the latter was used to prepare a working stock of each barcoded library at 1x107 molecules/μL (Table 1). Emulsion-based clonal amplification, bead washes and recovery, DNA library bead enrichment, and sequence primer annealing were carried out using the GS Junior Titanium emPCR (Lib-L) Kit and following manufacturer's protocols as outlined in the emPCR Amplification Method Manual (Lib-L)(v. April 2011). Enriched beads were prepared for sequencing on a GS Junior PicoTiterPlate Device using the GS Junior Titanium Sequencing Kit and following manufacturer's protocols as outlined in the Sequencing Method Manual (v. November 2011). Single-end massively parallel pyrosequencing was carried out in multiplex on a 454 GS Junior at the Sackler Institute for Comparative Genomics (American Museum of Natural History).