Ingestion of fluorescently labeled and phycoerythrin-containing prey by mixotrophic dinoflagellates

In order to experimentally investigate feeding by mixotrophic dinoflagellates, we developed protocols for the use of live protistan prey as markers of ingestion. CMFDA (5-chloromethyl-fluorescein diacetate), a vital green fluorescent stain, was used to label cultures of photosynthetic nanoflagellates, a diatom, and an oligotrichous ciliate. Cryptophytes were not readily stained with CMFDA, but phycoerythrin-containing members of this phylum have a distinct yellow-orange fluorescence and thus can be used unstained to demonstrate ingestion. With these complementary techniques, we qualitatively demonstrated feeding by the dinoflagellates Ceratium furca, Gymnodinium sanguineum, Gyrodinium estuariale, Prorocentrum minimum (= mariae-lebouriae) and Peridinium brevipes in natural assemblages from Chesapeake Bay, USA. We also used CMFDA-stained Isochrysis galbana (Prymnesiophyta) and unstained Cryptomonas sp. (Cryptophyta) in laboratory and field studies, respectively, to examine prevalence of feeding by G. estuariale as a function of prey density. However, determination of in situ grazing rates for mixotrophic dinoflagellates proved difficult, as only a small percentage of cells contained labeled food vacuoles following short incubations (less than or equal to 4 h) with stained prey added at tracer concentrations. The use oi CMFDA-stained cells and phycoerythrin-containing prey as markers of ingestion should also be applicable to species-specific feeding studies with other phagotrophic protists and micro-metazoa. The protocols presented here have advantages over the use of fluorescent microspheres or fluorescently labeled heat-killed algae (FLA) for investigating grazing or predation because many micrograzers do not readily ingest, or discriminate against, inert particles.