2.3. Actinomycin-D inhibition of development

Immediately after fertilization, embryos were transferred individually into the wells of 96-well plates with freshwater (0.4 ppt) and supplemented with concentrations of a transcriptase inhibitor, actinomycin-D (Sigma-Aldrich, St. Louis, MO), to examine the progression of development past the maternal-to-zygotic transition (MZT; Lee et al., 2014). Embryos that do not initiate genomic transcription in response to the inhibitor cannot transition morphological and in result can stall in development, allowing for the identification of which stage the MZT in this species (Podrabsky et al., 2017, Zamir et al., 1997). The final concentration of dimethyl sulfoxide (DMSO) was 1.0% v/v with 50, 100, 200, and 400 μM concentrations of actinomycin-D. An additional control group was included with 1.0% DMSO without the inhibitor. The morphological progression of actinomycin-D-treated embryos was compared to embryos under control conditions: freshwater and freshwater + 1.0% DMSO (n = 36 per treatment). Embryos were monitored and staged morphologically over a period of 50 h and the remaining individuals that survived treatments were also recorded.