Zooplankton samples were collected on NGA-LTER cruises along the Seward Line and within PWS during the summer, spring, and fall of 2018-20 aboard the R/V Sikuliaq, R/V Tiglax or the R/V Woldstad. During spring and summer cruises copepods were collected using a Quad net, a modified CalVet net (0.25 m mouth diameter, 1 m cylinder and 1.5 m cone), of 150-µm mesh hauled vertically from 100 m during the day, then live sorted and imaged. During summer 2019, animals were also collected during the night in the drogue net (0-200 m) of a towed Midi Multinet (0.25 m2) (Hydro-Bios, Germany), equipped with 500-µm mesh nets, and live sorted and imaged the following morning. The towed Multinet fished five discrete depth layers including: 0-20, 20-40, 40-60, 60-100, and 100-200 m. During summer 2020, we employed a vertically-hauled Midi Multinet of 150-µm mesh (fished during the day) for these same strata that were live-sorted and imaged immediately after collection to improve specimen quality. During summer and fall of all years at one or two deep-water Seward Line stations, and one or two PWS stations, we also fished the vertically-hauled Multinet at four additional depths: 200-300, 300-400, 400-600 and 600-1200 m (or 600-720 for PWS). A flowmeter installed in each net quantified the volume of water filtered. We targeted Neocalanus flemingeri, Neocalanus plumchrus and Neocalanus cristatus for sorting, staging, and imaging from each of these strata.
Up to 60 individuals of each stage for each species were analyzed, if present. The Neocalanus specimens were selected individually from homogenized temperature-regulated subsamples using a ladle or wide-bore plastic pipette. Only living, apparently undamaged individuals with intact lipid sacs were sorted and imaged using a Leica MZ16 microscope with either a Spot 4Mpx, Jenoptik 8Mpx, or Spot 12Mpx digital camera. Depending on stage and species, just prior to imaging three to fifteen animals were placed in a chilled embryo dish then water was reduced so animals shifted onto their sides (with gentle manipulation as needed). For every magnification setting, a calibration scale bar was added to the images. In cases where availability of animals was limited, animals with compromised lipid sacs were imaged to establish prosome length only. The calibrated images were later analyzed using Spot Software (V4.7 or 5.3) for determination of the prosome length (measured as maximum) and prosome width (measured from top of prosome to base of maxillipeds), the lipid sac length (measured from most anterior point to most posterior point), and lipid sac width (measured starting at the point closest to the base of the maxillipeds (ventral point) to most dorsal point). Animals that showed signs of compromised lipid sacs upon image inspection were only measured for prosome length. Adobe Photoshop (version CS6) was utilized to measure lipid sac area using the quick select tool to help demarcate the perimeter of the lipid sac. The number of pixels within the perimeter of the lipid sac was converted to area (mm2) using the pixel-to-µm calibration associated with each image.
To calculate lipid sac volume, we first assumed the lipid sac to be a cylinder. We used the measured lipid sac length and the measured lipid sac area to find the average diameter of the cylinder (i.e. area divided by length) in order to account for the varying height and shape of the lipid sac. We then used this average diameter to estimate the volume of the lipid sac. Body volume wa calcualted at a oblate spheroid from prosome length and prosome height.
Species List:
Scientfic_Name, Lifesciences Identifier (LSID)
Neocalanus flemingeri, urn:lsid:marinespecies.org:taxname:353708
Neocalanus plumchrus, urn:lsid:marinespecies.org:taxname:196772
Neocalanus cristatus, urn:lsid:marinespecies.org:taxname:104470
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