The hard clam is an important ecological and economic resource along the U.S. Eastern Seaboard. In Virginia alone, farm gate sales were estimated at $38.8 million in 2018 and $57.8 million in 2021, making Virginia the largest producer of hard clams in the U.S. This industry is primarily limited to higher salinity habitats on the seaside of the Eastern Shore of Virginia and lower Chesapeake Bay. Although the hard clam can be found in lower salinity habitats, they do not grow or survive at rates that are practical for productive aquaculture. Even in areas of higher salinity, hard clams are vulnerable to extreme precipitation events, which can lead to hyposaline stress and threaten natural and aquacultured populations. Transcriptomic analysis is a powerful tool for exploring the altered gene expression and cell cycle events that occur under osmotic stress. In the spring of 2019 and 2021, clam lines were created at the VIMS Eastern Shore Laboratory. Salinity exposures were conducted in the summer of 2021 with eight clam lines at four different salinities (35, 20, 15, and 12 ppt). RNA sequencing (RNA-Seq) data from either the gill (2019) or pooled whole bodies (2021) of exposed clams at 35 and 15 ppt were used to assess the transcriptomic response to low salinity stress. This study found 545 genes in the gills of adult hard clams and 465 genes in the whole bodies of juvenile hard clams that were significantly differentially expressed between 15 and 35 ppt. Some of the top genes by significance and log2 fold change were those in the categories of heat shock proteins, apoptosis, and cellular polarity. The amino acid sequences of these differentially expressed genes (DEGs) were assigned to key pathways like protein processing in the endoplasmic reticulum and Rap1 signaling. The response to 15 ppt was compared among clam lines, resulting in some interesting expression profiles. Some clam lines from the same population had a large number of genes differentially expressed in response to low salinity (15ppt), while other clam lines derived from different populations showed few differences. Some of the genes differentially expressed by different clam lines included heat shock proteins, inorganic ion regulators, and free amino acid isomerase enzymes. The observed difference between clam lines could indicate different tolerance to low salinity and different adapted molecular approaches to combat osmotic stress. This study identified key genes and pathways that could be the focus of future studies. Results from this study allow for a better understanding of how hard clams respond to low salinity stress and begin to lead to a determination of whether all populations of clams respond the same, which could benefit the aquaculture industry and lead to strategic breeding programs for enhanced low salinity tolerance in hard clams.

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