Transcriptomic Profiling of Differential Responses to Drought in Two Freshwater Mussel Species, the Giant Floater Pyganodon grandis and the Pondhorn Uniomerus tetralasmus Yupeng Luo 1,2. , Chao Li 1. , Andrew Gascho Landis 3 , Guiling Wang 2 , James Stoeckel 1 , Eric Peatman 1 * 1 School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, United States of America, 2 Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Agriculture, Shanghai, People’s Republic of China, 3 Georgia Department of Natural Resources, Wildlife Resources Division, Nongame Conservation Section, Social Circle, Georgia, United States of America Abstract The southeastern US has experienced recurrent drought during recent decades. Increasing demand for water, as precipitation decreases, exacerbates stress on the aquatic biota of the Southeast: a global hotspot for freshwater mussel, crayfish, and fish diversity. Freshwater unionid mussels are ideal candidates to study linkages between ecophysiological and behavioral responses to drought. Previous work on co-occurring mussel species suggests a coupling of physiology and behavior along a gradient ranging from intolerant species such as Pyganodon grandis (giant floater) that track receding waters and rarely burrow in the substrates to tolerant species such as Uniomerus tetralasmus (pondhorn) that rarely track receding waters, but readily burrow into the drying sediments. We utilized a next-generation sequencing-based RNA-Seq approach to examine heat/desiccation-induced transcriptomic profiles of these two species in order to identify linkages between patterns of gene expression, physiology and behavior. Sequencing produced over 425 million 100 bp reads. Using the de novo assembly package Trinity, we assembled the short reads into 321,250 contigs from giant floater (average length 835 bp) and 385,735 contigs from pondhorn (average length 929 bp). BLAST-based annotation and gene expression analysis revealed 2,832 differentially expressed genes in giant floater and 2,758 differentially expressed genes in pondhorn. Trancriptomic responses included changes in molecular chaperones, oxidative stress profiles, cell cycling, energy metabolism, immunity, and cytoskeletal rearrangements. Comparative analyses between species indicated significantly higher induction of molecular chaperones and cytoskeletal elements in the intolerant P. grandis as well as important differences in genes regulating apoptosis and immunity. Citation: Luo Y, Li C, Landis AG, Wang G, Stoeckel J, et al. (2014) Transcriptomic Profiling of Differential Responses to Drought in Two Freshwater Mussel Species, the Giant Floater Pyganodon grandis and the Pondhorn Uniomerus tetralasmus. PLoS ONE 9(2): e89481. doi:10.1371/journal.pone.0089481 Editor: Senjie Lin, University of Connecticut, United States of America Received August 6, 2013; Accepted January 22, 2014; Published February 25, 2014 Copyright: ß 2014 Luo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: peatmer@auburn.edu . These authors contributed equally to this work. Introduction As drought conditions recur with increasing frequency and severity in the Southeastern U.S., sessile aquatic organisms in freshwater ecosystems, particularly in streams, are bearing the brunt of these environmental perturbances [1–3]. Freshwater unionid mussel populations are already among the most endan- gered groups of organisms in the world [4]. Growing efforts to document the stunning diversity and varied life history strategies of unionids are also recording the measurable impacts of altered stream flows and thermal profiles on survival, recruitment, reproductive strategies, and community structure of these species [2,3,5–7]. The ability of individual species to tolerate drought conditions depends on several factors including severity and duration of the disturbance, stream habitat (debris, pools, etc.), and differences in behavioral and physiological responses to emersion/desiccation and heat stress [3]. Indeed, a recent study utilizing field and laboratory experiments revealed links between behavioral respons- es, physiological tolerances, and survival in three co-existing mussel species (Uniomerus tetralasmus (pondhorn); Pyganodon grandis (giant floater); and Lampsilis straminea (fatmucket)). There, the authors observed that a burrowing response in pondhorn was correlated with a higher tolerance to desiccation and higher survival (77%) compared to the water-tracking behavior and low tolerance and survival (0%) of giant floater [3]. The identification of underlying genetic mechanisms regulating these behavioral and physiological differences would provide key insights into adaptive responses of freshwater mussels to heat stress and drought. Previous studies in marine shellfish species have explored connections between variations in gene and protein expression and differences in latitudinal adaptation, differential success of native and invasive species, and resistance to summer mortality in the context of heat stress [8–13]. While such studies have traditionally required time-consuming and expensive gener- ation of molecular resources and have, therefore, been limited to a PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89481