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In an era of climate change, impacts on the marine environment include warming and ocean acidification. These effects can be amplified in shallow coastal regions where conditions often fluctuate widely. This type of environmental variation is potentially important for many nearshore species that are broadcast spawners, releasing eggs and sperm into the water column for fertilization. We conducted two experiments to investigate: 1) the impact of water temperature on sperm swimming characteristics and fertilization rate in sand dollars (Dendraster excentricus; temperatures 8-38°C) and sea urchins (Mesocentrotus franciscanus; temperatures 8-28°C) and; 2) the combined effects of multiple stressors (water temperature and pH) on these traits in sand dollars. We quantify thermal performance curves showing that sand dollar fertilization rates, sperm swimming velocities, and sperm motility display remarkably wide thermal breadths relative to red urchins, perhaps reflecting the wider range of water temperatures experienced by sand dollars at our field sites. For sand dollars, both temperature (8, 16, 24°C) and pH (7.1, 7.5, 7.9) affected fertilization but only temperature influenced sperm swimming velocity and motility. Although sperm velocities and fertilization were positively correlated, our fertilization kinetics model dramatically overestimated measured rates and this discrepancy was most pronounced under extreme temperature and pH conditions. Our results suggest that environmental stressors like temperature and pH likely impair aspects of the reproductive process beyond simple sperm swimming behavior. 

Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.