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Copepods were incubated with phytoplankton for 1 hour. Incubations were terminated by gently pouring the\u00a0contents of incubation bottles through a 53 mm sieve, then\u00a0rinsing the copepods into a petri dish with GF\/F filtered\u00a0water. A total of 12 copepods were sampled from each\u00a0stage for each phytoplankton and for the controls.<\/p>\n
Copepods were sorted under a dissecting microscope and\u00a0immediately transferred to glass microscope slides. Excess\u00a0water was removed with a fine-tipped Pasteur pipette to\u00a0immobilize the copepod for optimal viewing. To minimize\u00a0pigment degradation, slides containing the copepods were\u00a0transferred to the microscope as quickly as possible, always\u00a0within 2 min of sorting.<\/p>\n
A Nikon model E400 epifluorescence microscope at 20X magnification, outfitted with a Nikon B-2A long-pass filter cube (470EX\/515LP, Nikon) and a custom band-pass filter cube (430EX\/680EM, Omega Optical) was used. The longpass filter pair provided a brighter overall image than the band-pass filter pair and also fluoresced non-chlorophyll structures (e.g., the copepod exoskeleton fluoresces green with blue excitation). The band-pass filter cube showed fluorescence from only chlorophyll and phaeopigments. Photographs of the copepods were taken at each filter setting using a Canon Digital model T3i single-lens reflex camera (ISO 6400, F 1\/15) remotely controlled with the Canon Electro-Optical System Utility software to produce clear images of the epifluorescence (Vogt, 2013,Fig. 1).<\/p>\n
Images were processed with Photoshop CS6 (Adobe). Total gut area was manually digitized using the lasso and measurement tools on the long-pass-filtered images. The mean gut area of 12 individuals for each copepod species and stage was used for calculations. The threshold tool was used to select areas of the long-pass-filtered images containing pigment (gut pigment area) that exceeded baseline fluorescence. Signal intensity was not used in the calculation of the GPI (Gut Pigment Index) because pigment content and composition differed among phytoplankton taxa. Pixels of the band-pass-filtered images exceeding the threshold value were selected and their area estimated using the measurement tool. The gut-pigment area was then divided by the total gut area, yielding a relative GPI for each copepod. The same procedure was used to process images of copepods sampled from the control bottles. Copepods with <5% GPI were assumed not to have fed, as the GPI values in those samples were within the range of indices estimated from the controls.<\/p><\/div>","@type":"rdf:HTML"}],"http:\/\/ocean-data.org\/schema\/hasBriefDescription":[{"@value":"Pseudodiaptomus marinus gut area measured in pixels","@language":"en-US"}],"http:\/\/purl.org\/dc\/terms\/description":[{"@value":"
Adult and nauplius Pseudodiaptomus marinus<\/em> gut area were measured using epifluorescent microscopy.<\/p>\n Related Reference:<\/strong><\/p>\n Kamiyama, T. (2000). Application of a vital staining method to measure feeding rates of field ciliate assemblages on a harmful alga. Marine Ecology Progress Series<\/p>\n Li, A., et al. (1996). Ingestion of fluorescently labeled and phycoerythrin-containing prey by mixotrophic dinoflagellates.\" Aquatic Microbial Ecology 10(2): 139-147.<\/p>\n Merrell, J. R. and D. K. Stoecker (1998). Differential grazing on protozoan microplankton by developmental stages of the calanoid copepod Eurytemora affinis Poppe. Journal of Plankton Research 20(2): 289-304.<\/p>\n Vogt, R.A., T.R. Ignoffo, L.J. Sullivan, J. Herndon, J.H. Stillman, and W. Kimmerer. 2013. Feeding capabilities and limitations in the nauplii of two pelagic estuarine copepods, Pseudodiaptomus marinus<\/em> and Oithona davisae<\/em>. Limnology and Oceanography 58: 2145-2157<\/p><\/div>","@type":"rdf:HTML"}],"http:\/\/www.w3.org\/2000\/01\/rdf-schema#label":[{"@value":"4a_gut area-pixels","@type":"xsd:string"}],"http:\/\/ocean-data.org\/schema\/hasProcessingDescription":[{"@value":" BCO-DMO Processing:<\/strong>
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