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EFFECT OF ALTERED SALINITY ON LYTECHINUS VARIEGATUS DEVELOPMENT **

Abstract

Ocean salinity is affected by multiple factors associated with climate change, including changing patterns of rainfall and evaporation. Altered salinity changes carbonate availability in the ocean, which can disrupt development of organisms with calcium carbonate skeletons, like sea urchins. Lytechinus variegatus is a western-Atlantic species of sea urchin whose larvae exhibit unusual arm growth in response to altered salinity. However, the molecular basis of these skeletal abnormalities is not fully understood despite a well-known gene regulatory network for larval arm development. One important gene in this network is SM30, which encodes a key skeletal matrix (SM) protein required for building the larval skeleton. Altered salinity could impact its expression. We investigated this possibility by growing L. variegatus larvae for 72 hours post fertilization, the period for early arm growth, at three salinities (24, 27 [control], and 30 ppt). RNA was isolated from pooled samples of larvae and reverse transcribed to cDNA. We used published primers for a housekeeping gene, Setmar, and the biomineralization gene SM30. Both gave clear, single amplification products, which were confirmed by DNA sequencing. qPCR was then performed using SYBR green chemistry. Setmar amplified with 151% efficiency, and SM30 amplified with 97% efficiency. Due to the difference in these efficiencies, we used the Pfaffl analytical method to compare the relative level of SM30 expression, standardized to Setmar expression levels, across the different salinities. At the gastrula stage, 30 ppt showed a small increase in expression (1.6-fold), while 24 ppt had a larger increase (2.87-fold) in expression when compared to the control. At the two-arm stage, 30 ppt slightly decreased expression (0.94-fold), and 24 ppt had a small increase (1.76-fold). At the four-arm stage, 30 ppt showed a small increase (1.87-fold), and 24 ppt increased expression (2.63-fold). Based on these results, since SM30 expression normally changes during larval development as the skeleton forms, some of the differences we saw might be due to canonical growth rather than salinity effects. In general, however, our data may suggest that lower salinity (24 ppt) raises SM30 expression, but replication is needed to increase sample size for statistical analysis.

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