This project constituted an extension of our long-term microbial plankton time series in the San Pedro Channel, located roughly midway between Los Angeles and Santa Catalina Island, in open water about 20 km offshore (and depths from the surface to the bottom), to now cover 23 years of monthly observations. This latest grant also supported daily time series analyses from coastal water by the local Santa Monica Pier. The organisms we have been studying include bacteria, archaea, eukaryotes, and viruses, making this study unusually comprehensive and capable of discerning patterns not previously recognized. This is particularly valuable considering that this collection of microorganisms is responsible for the large majority of marine productivity and its consumption. Much of the work involved analyses of DNA (from the genomes of all the organisms, collectively called the metagenome) and RNA (transcripts of all the genes being expressed, also known as the metatranscriptome). As part of this study we validated our community composition analyses with carefully curated mock communities. Among the intellectually meritorious outcomes were observations that the bacterial and archaeal communities were much more stable over months and years than were the eukaryotic communities, though both showed seasonal patterns. The eukaryotic phytoplankton (producers) were more stable than eukaryotic heterotrophs (consumers). Bacteria and archaea made up a surprisingly large proportion of the organisms in the size fraction we had thought would be more dominated by protists (they constituted 40-90% of the ribosomal RNA marker genes in the 1 micrometer size fraction). We also determined that during the warm El Nino years 2014-2015, there was a community shift where the summer microbial community occurred year-round instead of having the usual shift to a winter community in that season. Interestingly, during that warm period, a particular type of Prochlorococcus cyanobacterium appeared at our study site, having not been seen there over the decade before, but one that usually lives in tropical areas. These shifts in communities provide direct evidence of what kinds of changes to expect in a warming world. Our studies also determined previously unrecognized diversity patterns with ocean depth, notably that prokaryotic (bacterial and archaeal) diversity peaked at mid-depths while the protistan diversity peaked at the surface. In studies of microbial interactions, we found that DNA viruses were particularly important in controlling the free-living bacterial community composition while phytoplankton were most important in controlling the particle-attached bacterial community. Our daily time series studies at Santa Monica Pier provided major insights into the conditions that lead to different kinds of algal blooms, in that we were lucky to observe blooms of both diatoms and dinoflagellates, the two guilds of phytoplankton that yield most large algal blooms globally. In metatranscriptional time series studies, we found that diatoms, which bloomed following upwelling events, expressed an array of gene categories related to dissolved inorganic nitrogen utilization, and genes related to the catabolism of chitin that may have prolonged their bloom duration following nitrogen depletion. Conversely, dinoflagellates bloomed under less-replete inorganic nitrogen conditions, exhibited less variation in transcriptional activity, and expressed few gene categories associated with dissolved inorganic nutrients during their bloom. Dinoflagellate profiles exhibited evidence of proteolysis and heterotrophy that may have enabled them to circumvent nutrient limitation and bloom to high abundances. Taken together, diatom and dinoflagellate transcriptional profiles illustrated guild-specific physiology, tuned to respond to and thrive under distinct environmental windows of opportunity. Broader impacts of the study include showcasing our work on our websites as well as supporting a significant education and training component of several early-career marine scientists (PhD students, Masters students, technicians), undergraduate students (from USC and elsewhere as part of a REU program) and students from Los-Angeles area high schools and colleges. Last Modified: 11/22/2023 Submitted by: JedAFuhrman