From Jungbluth et al. 2017 – MEPS:

A series of 5 bottle incubation experiments (hereafter referred to as E1 to E5; see Table 1) were conducted over a 10 d period (27 May to 5 June 2013) to measure grazing by copepod nauplii on the natural prey assemblage collected from Stn S3, located in the southern semi-enclosed basin of Kane‘ohe Bay, Oahu, Hawaii (21° 25’ 56" N, 157° 46’ 47"W; Jungbluth & Lenz 2013). The copepods were N3 and N4 stage nauplii of Parvocalanus crassirostris and Bestiolina similis. Concurrent experiments were run to measure microzooplankton community grazing, and to quantify in situ predator and prey abundan ces. The results from these experiments were used to correct for multiple trophic interactions within the bottle incubations, as these interactions can mask the effect of metazoan grazing (Nejstgaard et al. 2001). Salinity and temperature in the field were measured using a YSI 6600V2 sonde prior to collecting water for bottle incubations. Daily rainfall estimates were obtained from a rain gauge located at Luluku (www.prh.noaa.gov), and weather station data from the Hawaii Institute of Marine Biology (HIMB) (www.himb.hawaii.edu/weatherstation/) were used for estimates of the wind magnitude, wind direction, and solar irradiance.

Copepod nauplii used in the incubations were obtained from laboratory culture populations of P. crassirostris and B. similis established from animals previously collected in Kane‘ohe Bay (P. Lenz lab). Both species are capable of completing naupliar development in less than 3 d and reaching the adult stage (C6) in approximately 7 to 8 d (McKinnon et al. 2003, VanderLugt et al. 2009). Use of these monospecific cultures enabled us to produce high abundance naupliar cohorts of a specific age for grazing incubations. To produce these cohorts, adults of each species were isolated and fed 1 × 106 cells ml-1 Tisochrysis lutea (formerly Isochrysis galbana Tahitian strain; Bendif et al. 2013) 18 h prior to the start of each experiment to increase naupliar production. The adults were removed 6 h later, resulting in a cohort of nauplii (N3 and N4; note that N3 are the first feeding stage of these nauplii) raised at the experimental temperature of 21°C by the beginning of each experiment. Sets of ~50 nauplii were isolated into small volumes (<10 ml) of 0.2 µm filtered seawater and held for 1 to 3 h prior to the start of each grazing experiment. This procedure resulted in minimal exposure of the N3 and N4 nauplii to prey prior to the start of the grazing experiments. Seawater for the prey assemblage was collected from 2 m depth using a 5 l General Oceanics Niskin bottle deployed by hand line, with the contents gently added (silicone tubing) to two 20 l polycarbonate carboys.

Separate experiments indicated that longer incubation times decreased ingestion estimates within the grazing treatments, likely due to the fast development rates of our nauplii (< 24 h inter-molt period) and the diverse and rapidly changing prey community in this relatively warm (> 20°C) system (Jungbluth et al. 2017). Thus, 6 h incubations were chosen to give the most representative view of naupliar grazing rates on natural prey, with conditions closest to those in situ, and in order to minimize nutrient remineralization and other food web interaction effects that can be significant during longer incubations (Roman & Rublee 1980).

Grazing incubations were performed in pre-washed (10% HCl rinse, followed by 3 rinses with ambient 0.2 µm seawater) polycarbonate bottles (total volume: 1120 ml) with 35 µm gently pre-screened bulk seawater offered as prey. It is possible that our nauplii would consume prey > 35 µm given the opportunity, however the small size of the copepod species in our study (~40 µm wide, ~70 µm long; P. crassirostris N1 dimensions) necessitated the re moval of prey > 35 µm to ensure removal of other nauplii from the field. Our initial expectations were that the optimum prey size for our species would be 2 to 7 µm (Berggreen et al. 1988, Hansen et al. 1994), therefore the prey included here (< 35 µm) should represent a majority consumed naturally by our species.

The experimental nauplii were transferred into the 1120 ml grazing bottles at 2 densities (42-51 [moderate] and 81-95 [high] nauplii; see Table 1) and placed on a bottle roller (Wheaton) at 5 rpm in the dark for 6 h. The 2 nauplius densities were tested to ensure that we could detect removal of prey cells relative to controls over our incubation period, since a predator density that is too low may result in insignificant prey removal relative to control bottles. Removal of cells in treatments relative to control bottles was detected in the moderate density treatments and ingestion rate estimates were comparable to those from higher density bottles. Since results were comparable between moderate and high density treatments, results reported here focus on bottles with ~50 nauplii l-1, also because treatment replication was better with moderate density bottles (n = 3 per experiment) than high density bottles (n = 2). This density of nauplii is well within the range of total nauplius concentrations reported in previous studies in Kane‘ohe Bay (7 to 68 total nauplii l-1; Hoover et al. 2006) and within the range of each species abundance we have previously measured following storm runoff events in the bay (M.J.J. pers. obs.).

Treatment bottles were run in triplicate, with 2 or 3 no-nauplii control bottles for each experiment (2 for E1 to E2, 3 for E3 to E5). Experiments were incubated at 21°C, which is within the range of the annual temperature fluctuation in Kane‘ohe Bay (20 to 29°C during the previous 5 yr; HIMB weather data). No nutrients were added to the bottles, because controls and experimental bottles were considered approximately equally influenced by nitrogen remineralization from grazing processes, due to the presence of other < 35 µm microzooplankton grazers in all bottles and the short incubation times (6 h). Nauplii are known to have low expected nitrogen remineralization (~10- fold lower than adults) due to their small biomass compared to adults (Vidal & Whitledge 1982, Mauchline 1998); at 50 nauplii l-1, excretion rates were estimated to be 2 to 3 orders of magnitude below the in situ average nitrogen concentrations in Kane’ohe Bay (0.2 to 1.0 µM; Drupp et al. 2011). Initial and final time-point measurements included samples to quantify particle size and abundance in the 2-35 µm size range from the Coulter counter (CC), as well as samples for specific prey types, including chlorophyll a (chl a) and the abundance and biomass of types of nano- and microplankton. Prey types and CC-quantified potential prey were not expected to be equal; some prey types include cells < 2 µm, while the lower limit of the CC was 2 µm. Nauplii were re covered at the end of the experiments to check their condition (alive/dead; no dead nauplii were found), then preserved in 10% paraformaldehyde, stained with 1% Rose Bengal, and enumerated using microscopy for use in clearance and ingestion rate estimates.

For complete methodology, see the Supplemental Files section.