The conclusions of this study were complicated by examination of multiple Esoptrodinium isolates that differed from one another in a specific trait (possession of pigmented plastids) that would be expected to influence the experimental results (capacity for mixotrophy). All Esoptrodinium isolates required food cells to grow, demonstrating obligate phagotrophy. The tested isolate with obvious Enzalutamide in vitro pigmented chloroplasts (UNCCP) contained detectable chlorophyll and exhibited a positive biomass response to light in the absence of

food, demonstrating phototrophy (and thus the capacity for mixotrophy). Furthermore, this isolate appeared to require light for sustained growth, even when saturating abundances of fresh prey cells were provided daily. These characteristics suggest what could be functionally categorized as obligate mixotrophy, a rarely demonstrated nutritional strategy in dinoflagellates (Stoecker 1998). However, the observed responses of the tested Esoptrodinium isolates that contained either cryptic, barely visible plastids (isolate RP) or no visible plastids (isolate HP) complicate this interpretation. Both of these essentially “colorless” (not counting the eyespot) isolates lacked chlorophyll as a plainly visible or microfluorimetrically CH5424802 detectable cell component, and not surprisingly did not appear capable of phototrophic biomass production (i.e., not mixotrophic). Nevertheless, they appeared to require light for sustained

growth, even when saturating abundances of fresh prey cells were provided daily. This physiological response is classified here as photoobligate but nonphototrophic. The potential reason for the observed requirement for light among nonphototrophic Esoptrodinium isolates remains unknown

(below), but calls into question the possibility that the pigmented chloroplast-bearing isolate (and by extension others like it) may also be photoobligate rather than obligately phototrophic. It also remains possible that the Esoptrodinium isolates were capable of sustained growth in darkness, but at such a reduced rate as to be unappreciated in these experiments. Likewise, other unknown culture conditions (e.g., presentation of prey species not tested here) might permit significant growth of the dinoflagellates in darkness. Regardless, all repeated attempts to serially cultivate Esoptrodinium Calpain isolates with food in darkness have quickly failed, whereas the same strains have been maintained with food in light (12:12 L:D cycle) for >3 years at the time of this writing. These observations further suggest light is required for sustained growth of Esoptrodinium, either directly or indirectly as mediated through microalgal prey. Several alternative, nonmutually exclusive hypotheses could explain the observed requirement for light by Esoptrodinium, even among isolates that appear to lack chlorophyll. First, light may be required for proper digestion of prey.