Trophic changes in d15N of AAs from phytoplankton to protistan and metazoan plankton consumers were examined in six chemostat set-ups, including five two-stage experiments and one 3-stage experiment (Décima et al. 2017).

All experiments were conducted in a temperature-controlled room (16 or 18 degrees Celsius) with the algal prey in 2.2-L stage 1 reactors (dilution rate = 0.5 d-1) receiving continuous nutrient input from a 20-L reservoir under continuous light (~120 umol photon m-2 s-1). Seawater collected from the Scripps pier (La Jolla, CA) was autoclaved and filtered for all media preparations, using f/2 Guillard concentrations for all nutrients except NO-3 or PO3- 4, which were varied to produce conditions of N- (Exps. 1 and 4-6) or P-limitation (Exps. 2), while Exp. 3 had no imposed limitation. Flow between the nutrient reservoir and the chemostat culture reactors was continuously pumped through platinum-cured silicone tubes using a Masterflex L/S model 7519-05 peristaltic pump equipped with individually adjustable cassettes for each tube.

For the two-stage experiments, consumers in the stage 2 reactors were grown on the phytoplankton from stage 1 outflow under continuous light or continuous dark conditions to either allow (light) or suppress (dark) phytoplankton uptake of the nutrients regenerated by consumer excretion. The reactors were monitored daily for 12-15 days to ensure constant conditions, thus minimizing predator: prey isotopic mismatch, and samples were taken for cell abundances and biovolumes using an Elzone counter and epifluorescence microscopy. Samples for CSIA-AA were collected at the end of the experiments on pre-combusted 47-mm GFF filters, stored at -80 C, and dried at 60 C for at least 24 h before analyses.

For the 3 stage experiment, the second and third stage reactors had 3.6 L volumes. In the third reactor, metazoan consumers were grown on protistan zooplankton flowing from the second reactor, in turn growing on phytoplankton from the first reactor. Experiments were conducted for 14 days, after which the first and second stages were harvested by filtering the contents onto pre-combusted 47-mm GFFs. Metazoan consumers and <200-um particles in the third-stage reactor were collected as above.

Samples were hydrolyzed and purified AAs derivitized according to established protocols (Popp et al. 2007; Hannides et al. 2009; Décima et al. 2017). All samples were injected (splitless injector) onto a forte BPx5 capillary column (60 m x 0.32 mm x 1.0-um film thickness) at an injector temperature of 250 C with a constant helium flow rate of 1.4 ml min–1. The column was initially held at 50 C for 2 min and then increased to 125 C at a rate of 15 C per min. Once at 125 C, the temperature was increased at a rate of 3 C per min to 160 C and then to 190 C at a rate of 4 C per min. The final temperature of 300 C was reached by ramping to 275 C at 6 C per min and then 15 C per min afterward. Samples were analyzed in triplicate and normalized to the known δ15N values of a suite of 14 AAs analyzed before and after each set of 3 samples.

References:
Décima, M., M. R. Landry, C. J. Bradley, and M. L. Fogel. 2017. Alanine d15N trophic fractionation in heterotrophic protists Limnol. Oceanogr., doi:10.1002/lno.10567

Gutiérrez-Rodríguez, A., Décima, M., Popp, B. N., and Landry, M. R. 2014. Isotopic invisibility of protozoan trophic steps in marine food webs. Limnol. Oceanogr. 59: 1590-1598.

Hannides, C. C. S., B. N. Popp, M. R. Landry, and B. S. Graham. 2009. Quantification of zooplankton trophic position in the North Pacific Subtropical Gyre using stable nitrogen isotopes. Limnol. Oceanogr. 54: 50-61.

Landry, M.R. and M.R. Décima. 2017. Protistan microzooplankton and the trophic position of tuna: Quantifying the trophic link between micro- and mesozooplankton in marine food webs. ICES J. Mar. Sci. doi:10.1093/icesjms/fsx006

Popp, B. N., B. S. Graham, R. J. Olson, C. C. S. Hannides, M. J. Lott, G. A. Lopez-Ibarra, F. Galvan-Magana, and B. Fry. 2007. Insight into the trophic ecology of yellowfin tuna, Thunnus albacares, from compound-specific nitrogen isotope analysis of proteinaceous amino acids, p. 173-190. In T. E. Dawson and R. T. W. Siegwolf [eds.], Stable isotopes as indicators of ecological change. Terrestrial Ecology Series. Elsevier Academic Press.

AA_d15N.csv