From Jungbluth et al. 2017 – MEPS:

Estimates of in situ naupliar abundance

Naupliar abundances of the 2 target species in situ were estimated using a quantitative polymerase chain reaction (qPCR)-based method (Jungbluth et al. 2013), as well as microscopic counts of calanoid and cyclopoid nauplii. The qPCR-based method allows application of individual species grazing rates to in situ abundances to estimate the total potential grazing impact of each species. Samples were collected by duplicate vertical microplankton net tows (0.5 m diameter ring net, 63 µm mesh) from near bottom (10 m depth) to the surface with a low speed flow meter (General Oceanics). The contents of each net were split quantitatively. One half was size-fractionated through a series of 5 Nitex sieves (63, 75, 80, 100, and 123 µm) to separate size groups of nauplii from later developmental stages, and each was preserved in 95% non-denatured ethyl alcohol (EtOH). The second half of the sample was preserved immediately in 95% EtOH for counts of total calanoid and total cyclopoid nauplii, which were used for comparison to the qPCR-based results of the abundance of each calanoid species. All samples were stored on ice in the field until being transferred to a -20°C freezer in the laboratory. EtOH in the sample bottles was replaced with fresh EtOH within 12 to 24 h of collection to ensure high-quality DNA for analysis (Bucklin 2000).

The 3 smallest plankton size fractions from the net collection were analyzed with qPCR to enumerate P. crassirostris and B. similis nauplius abundances (Jungbluth et al. 2013). In brief, DNA was extracted from 3 plankton size fractions (63, 75, and 80 µm) using a modified QIAamp Mini Kit procedure (Qiagen). The total number of DNA copies in each sample was then measured using species-specific DNA primers and qPCR protocols (Jungbluth et al. 2013). On each qPCR plate, 4 to 5 standards spanning 4 to 5 orders of magnitude in DNA copy number were run along with the 2 biological replicates of a size fraction for each sampling date along with a no template control (NTC), all in triplicate. A range of 0.04 to 1 ng µl-1 of total DNA per sample was measured on each plate ensuring that the range of standards encompassed the amplification range of samples, with equal total DNA concentrations run in each well on individual plates. In all cases, amplification efficiencies ranged from 92 to 102%, and melt-curves indicated amplification of only the target species. The qPCR estimate of each species' mitochondrial cytochrome oxidase c subunit I (COI) DNA copy number was converted to an estimate of nauplius abundance using methods described in Jungbluth et al. (2013).

Conditions

Salinity and temperature in the field were measured using a YSI 6600V2 sonde prior to collecting water for bottle incubations. For chl a, triplicate 305 ml samples were filtered onto GF/Fs (Whatman), flash-frozen (LN2), and kept at -80°C freezer until measurements were made 4 mo later. Chl a (and phaeopigment) was measured using a Turner Designs (model 10AU) fluorometer, using the standard extraction and acidification technique (Yentsch & Menzel 1963, Strickland & Parsons 1972).

For complete methodology, see the Supplemental Files section.