Unionidae Transcriptome Project(UTP). Unionidae

Specimens of the endangered Alasmidonta heterodon were collected from the Connecticut River. Alamidonta varicosa were sampled from Croyden Brook, a tributary of the Connecticut River. Elliptio complanata were sampled from Pine Creek, a tributary of the Susquehanna River. RNA was isolated from 50-100 mg of fresh or RNA Later- preserved gill, mantle, foot, and hepatopancreas tissue from each of five specimens. The tissues were subjected to a Trizol extraction following methods described by P. Chomczynski and N. Sacchi, Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction (Analytical Biochemistry, vol. 162, p.156). Removal of ribosomal RNA was performed using Lock-Nucleic Acids (Invitrogen RiboMinus Eukaryote Kit for RNA-Seq #A10837-08). The cleaned mRNAs from each species were subjected to quantification and qualification using a Bioanalyzer 2100 Lab-on-a-Chip (Agilent Technologies). mRNAs for each species were sent to the University of Iowa DNA Core Sequencing Facility for cDNA library preparation using the cDNA Rapid Library Preparation Method for GS FLX Titanium Series (October 2009). 200ng of sample RNA was fragmented and then prepared for double- stranded cDNA Synthesis using the cDNA Synthesis System Kit (Roche) followed by fragment end repair using New England Biolabs NEBNext End Repair Module. The RL Multiplex IDentifiers (MID) Adaptor was ligated to the sample following end repair. Small fragment removal was performed using AMPure Beads (Agencourt). Each library was quantified and then prepared for emulsion PCR. Unionids, or freshwater mussels, constitute one of the most sensitive and rapidly declining faunal groups in the world. Mussels provide critical ecosystem services by filtering thousands of liters of water a day, enhancing stream bed stability, increasing sediment oxygen content, and serving as a link in aquatic feeding webs. Unfortunately, because they are filter feeders with a sedentary adult phase, they are susceptible to habitat alteration and water pollution; nearly 70% of North American freshwater mussel species are already extinct or vulnerable to extinction. Because little is known about their physiology, our transcriptome project is aimed at identifying genes mediating adaptation and conferring resiliency in the face of ecological challenge. Identification of genes and proteins relevant to adaptation will contribute to conservation and restoration efforts.