Zooplankton was collected from 31°N to 29°N aboard the R/V Savannah at the 25m and 40m isobaths using a 1M (mouth opening) 5M length 200 µm mesh cone plankton net equipped with a filtering (202 µm mesh) cod-end.  The mouth of the net was mounted in a swivel towing frame that allowed the opening of the net to track the water current. A mechanical flow meter (General Oceanics Part# 2030RC) was mounted in the center of the net. Flowmeter measurements for 2015 cruises (pdf) The net was deployed from a gently drifting ship and the entire water column was sampled by lowering the net to near the bottom and retrieving it (oblique tow).  Net speed was maintained at approximately 15 meters per minute. Once retrieved the contents of the net were collected on a 200 µm sieve and preserved in 60% non-denatured ethanol.

Samples were split twice to create four subsamples using a Folsom plankton splitter.

One of the subsamples was diluted to a known volume and stirred gently. Dilutions were adjusted depending on the density of zooplankton so that sufficient numbers of species were present in each sample to obtain a reliable estimate of the major taxonomic groups in each sample. Replicate aliquots from the subsample were counted under a dissecting microscope using a Bogorov counting chamber. The volume of sample counted was adjusted based on the density of zooplankton present but was generally 5 ml. A Stempel pipette was used to dispense the counted volume.

In the case where the identity was uncertain, representative examples of the unidentified zooplankton were identified by DNA barcoding.  DNA extractions were performed using the DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer’s protocol except that samples were macerated and homogenized with Kimble/Kontes Cordless Motor, the proteinase K incubation was extended to ~24 hours (overnight) and the volume of elution buffer AE varied depending on sample size (60ul for most). For DNA quantitation and quality testing, one (1) ul of DNA was used to measure the 260/280 ratio using the Thermo Scientific™  NanoDrop Lite Spectrophotometer.  The presence of high molecular weight DNA in each extract was visually confirmed by agarose (1%) gel electrophoresis.  Good quality DNA extractions, inferred by 260/280 ratio and electrophoresis, were selected for amplification by Polymerase Chain Reaction (PCR) of a fragment of the mitochondrial cytochrome oxidase subunit I (mtCOI) gene.  A 708 base-pair mtCOI gene fragment was amplified using the consensus primer pairs LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994).  Amplification was facilitated using a Bio-Rad T100Thermocycler with GoTaq Hot Start Green Master Mix. Cycling conditions were: 10 min at 95 °C; 35 cycles of 30 sec at 95 °C, 1 min at 47 °C and 1 min at 72 °C; and 10 min at 72 °C. PCR product quality and quantity was assessed by 2% agarose gel electrophoresis and subsequently sequenced by Eurofins Genomics. DNA sequences obtained were compared to a reference database library using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990) available at the National Center for Biotechnology Information (NCBI) and Bold Systems Identification engine for CO1 gene.