Award: OCE-1756767

This collaborative project investigated the physiological ecology of marine copepods in the sub-arctic Pacific. The Gulf of Alaska sustains major fisheries that depend directly or indirectly on small planktonic organisms, zooplankton, that inhabit the pelagic ecosystems. The annual spring algal bloom is an important source of energy for zooplankton including four co-occurring lipid-rich copepod species (Neocalanus flemingeri, Neocalanus plumchrus, Neocalanus cristatus and Calanus marshallae). The copepods' high-lipid content makes them an important food source for higher trophic levels, including larval fishes. Like fat on a hibernating bear, the copepods store lipid in preparation for a seasonal dormancy, termed "diapause." By ending dormancy at the right time females can synchronize reproduction with the next season's algal bloom. This project was motivated by the need to understand how the timing and magnitude of spring algal blooms affect the physiological state of copepods preparing for diapause. Physiological profiles of individual copepods were generated by measuring, for each gene, its "expression level" -- the amount of messenger RNA "transcript" (mRNA) each produces. The project had outcomes in four areas: effects of food on diapause preparation; impact of a variable environment on physiological profiles; cross-species profiling, and evaluating reproductive potential. [1.] The effects of food quantity and quality on lipid accumulation, development and gene expression in laboratory experiments on juvenile and pre-adult N. flemingeri revealed an exceptional adjustment in physiology during low food conditions. It included lowering metabolic rate while maintaining accumulated lipid. This was also observed in field-collected individuals during three successive years, 2015, 2016 and 2017, a period during and just after the marine heatwave of 2014-2016. Gene expression profiling detected signs of food stress in 2016, similar to those seen in the laboratory experiments, and coinciding with unusually low spring algal levels. [2.] The ability of copepod species to persist in the northern marine environments in the face of environmental change (global warming, ocean acidification, marine heatwaves) will depend on their capacity for physiological acclimatization. The gene expression responses to different food levels demonstrate physiological plasticity that allows the copepods to succeed in an environment with steep gradients in food quantity and quality. In 2015, comparisons of individuals collected from offshore stations identified a signature stress response, probably due to local toxins. [3] The project generated molecular resources for five additional species, for use in cross-species profiling. These included lipid-rich N. plumchrus, N. cristatus and Calanus marshallae, and two other large copepods, Eucalanus bungii and Metridia pacifica. These resources are needed for multi-species analyses using meta-transcriptomics in comparisons among multiple co-occurring species. New bioinformatic workflows were used to develop a common analysis toolkit for application to existing and future data sets. Because of the inherent complexity of RNA Seq data (10,000s of transcripts) a dimensionality-reducing tool was adapted from artificial intelligence developers: t-distributed Stochastic Neighbor Embedding (t-SNE). This tool generates visually-compelling two-dimensional clustering of expression profiles that share significant similarities. It was tested in a workflow designed to distinguish the signatures of a North Atlantic copepod (Calanus finmarchicus) on a diapause track (with reproduction occurring in the following spring) from one programmed to develop directly without diapausing (with reproduction in the current season). The workflow was used to construct six "filters" consisting of subsets of genes designed to differentiate between the two developmental programs. Two of these performed particularly well. [4] Neocalanus flemingeri females awakening from diapause enter the reproductive program, which takes 7-8 weeks before mature eggs are released. This was monitored with a histochemical technique adapted from other crustacean studies. Egg-precursor cells ("oogonia" and "oocytes") were labeled florescently by incubation in 5-Ethynyl-2-deoxyuridine (EdU). The technique allows visualization of cells duplicating their DNA (the "S-phase" of the cell-division cycle), the first step in "oogenesis," the production of yolk-provisioned eggs (mature oocytes). Fluorescent images quantified by confocal microscopy revealed that the DNA-duplication started within 24 hours of diapause termination and stopped after four weeks. This significant finding suggests that the limited period for oocyte production assures availability of adequate maternal resources for subsequent full provisioning of mature eggs. Education/outreach was integrated with the research. Two post-doctoral fellows, three graduate students, four undergraduate and two post-baccalaureate students were trained in biological oceanography, and zooplankton molecular and field studies. One female student progressed from undergraduate assistant to graduate student during the COVID pandemic, a period of limited opportunities for students in transition. Because fishing is a major industry in the Gulf of Alaska, outreach focus was in Alaska. Student team members were trained in outreach, and participated in teacher workshops, laboratory tours for K-12 and other educators, and the Tsunami Bowl in Homer and Seward, Alaska in 2019, 2021 and 2023. Project participants worked with childrens book author Christy Peterson and Whale Times, Inc to create an e-Book "Seasons of the Sea" describing seasonality of marine life in the Gulf of Alaska. Last Modified: 12/30/2023 Submitted by: DanielKHartline