DOI: 10.5061/dryad.sqv9s4n1c
title: Data from: A high-throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia

description: Food ingestion is one of the most basic features of all organisms.
 However, obtaining precise — and high-throughput — estimates of feeding
 rates remains challenging, particularly for small, aquatic herbivores such
 as zooplankton, snails, and tadpoles. These animals typically consume low
 volumes of food that are time consuming to accurately measure. We extend a
 standard high-throughput fluorometry technique, which uses a microplate
 reader and 96-well plates, as a practical tool for studies in ecology,
 evolution, and disease biology. We outline technical and methodological
 details to optimize quantification of individual feeding rates, improve
 accuracy, and minimize sampling error. This high-throughput assay offers
 several advantages over previous methods, including: i) substantially
 reduced time allotments per sample to facilitate larger, more efficient
 experiments; ii) technical replicates; and iii) conversion of in vivo
 measurements to units of carbon (mg dw L-1), which enables broad-scale
 comparisons across an array of taxa and studies. To evaluate the accuracy
 and feasibility of our approach, we use the zooplankton, Daphnia dentifera
 as a case study. Our results indicate that this procedure accurately
 quantifies feeding rates and highlights differences among seven genotypes.
 The method detailed here has broad applicability to a diverse array of
 aquatic taxa, their food, environmental contaminants (e.g., plastics), and
 infectious agents. We discuss simple extensions to quantify
 epidemiologically relevant traits, such as pathogen exposure and
 transmission rates, for infectious agents with oral or trophic
 transmission.
description: This method uses a microplate reader (TecanÓ, Maennedorf,
 Switzlerand) to quantify feeding rates using <i>in vivo</i>
 narrow-band fluorometry, a standard and widely-used method for accurately
 measuring chlorophyll-<i>a</i>  (Lorenzen, 1966; Kalaji et
 al., 2014). In brief, the goal is to compare the fluorescence of algae in
 tubes with animals (consumers) vs. the fluorescence of algae in the
 animal-free (consumer-free) controls, following Sarnelle and Wilson
 (2008). We provide a detailed protocol aimed at improving repeatability
 and analytical accuracy, while minimizing variation among samples. The
 most important, but easily overlooked, details include: (i) preparing all
 media in batch cultures and mixing it continuously prior to and throughout
 distribution to each biological replicate; (ii) conducting the assay under
 minimal light conditions to prevent spurious spikes in fluorescence; (iii)
 ensuring that the ratio of chlorophyll to carbon remains constant across
 assays; and (iv) pair-matching plate-specific controls with their
 respective treatment samples to reduce among-plate variation.
description: To minimize noise introduced by among-read variation (Petersen
 & Nguyen, 2005; Resch-Genger et al., 2005), it is crucial that
 each plate is treated as a block and contains control samples (i.e., algae
 from no-animals/consumers replicates) and that the average of these
 plate-specific controls are used to calculate feeding rates. That is, in
 the equations below, the control values come from each plate and are not
 averaged across all controls. This design reflects a matched-pairs layout
 and uses replication within blocks to help tease apart main effects, block
 effects, and their interaction (Gotelli & Ellison, 2004). This
 added step is an obvious limitation of this method. However, this is
 currently the best solution for maximizing the signal-to-noise ratio given
 the extreme sensitivity of modern fluorometers. In the online
 supplementary material that accompanies this manuscript, we include R code
 to facilitate these additional quality-control steps.