DOI: 10.5061/dryad.kwh70rz0p
title: Inter- and intraspecific differences in rotifer fatty acid composition during acclimation to low quality food

description: Biochemical food quality constraints affect the performance of consumers
 and mediate trait variation among and within consumer species. To assess
 inter- and intraspecific differences in fatty acid retention and
 conversion in freshwater rotifers, we provided four strains of two closely
 related rotifer species, Brachionus calyciflorus sensu stricto and
 Brachionus fernandoi, with food algae differing in their fatty acid
 composition. The rotifers grazed for five days on either Nannochloropsis
 limnetica or Monoraphidium minutum, two food algae with distinct
 polyunsaturated fatty acid (PUFA) profiles, before the diets were switched
 to PUFA-free Synechococcus elongatus, which was provided for three more
 days. We found between- and within-species differences in rotifer fatty
 acid compositions on the respective food sources and, in particular,
 highly specific acclimation reactions to the PUFA-free diet. The different
 reactions indicate inter- but also intraspecific differences in
 physiological traits, such as PUFA retention, allocation, and
 bioconversion capacities, within the genus Brachionus that are most likely
 accompanied by differences in their nutritional demands. Our data suggest
 that biochemical food quality constraints act differently on traits of
 closely related species and of strains of a particular species and thus
 might be involved in shaping ecological interactions and evolutionary
 processes.
description: <i>Cultivation of
 food organisms</i> The phytoplankton species
 <i>Synechococcus elongatus</i> (SAG 89.79, Culture Collection
 of Algae, University of Göttingen, Germany), <i>Monoraphidium
 minutum</i> (SAG 243-1) and <i>Nannochloropsis
 limnetica</i> (SAG 18.99) were used as food in the experiments
 because they differ in their fatty acid composition (figure 1). Each
 species was cultured in a chemostat system filled with 700 ml of sterile
 and vitamin-supplemented Woods Hole Culture Medium (WC). They were
 cultured with a flow rate of 0.3 d<sup>-1</sup> at 22 °C under
 continuous illumination of 100 µmol photons m<sup>-2
 </sup>s<sup>-1</sup>. At the beginning, middle, and end
 of the experiments, subsamples were taken from each algal chemostat
 culture and analysed for particulate organic carbon (POC) and fatty acids.
 Therefore, algae were collected on glass-fibre filters (Whatman GF/F, 25
 mm diameter) with &gt;0.15 mg C for carbon analysis and &gt;0.5 mg
 C for fatty acid analysis; carbon concentrations were estimated from
 pre-established light extinction-carbon regressions (800 nm; UV Shimadzu
 spectrophotometer, Duisburg, Germany).  <i>Cultivation of
 rotifers</i> Three strains of <i>B.
 calyciflorus</i> s.s. (‘IGB’, ‘Cornell’, ‘USA’) and one strain of
 the closely related species <i>B. fernandoi</i> (‘No.2484’)
 were used in the experiments (see supplementary material 1 for strain
 information and Michaloudi <i>et al.</i> (2018) and
 Paraskevopoulou <i>et al.</i> (2018) for species
 identification). Rotifers were precultured in bottles with sterile WC and
 <i>M. minutum</i> (SAG 243-1) as food. On a daily basis, food
 concentrations were estimated from aliquots taken from each replicate
 using algae-specific pre-established light extinction-carbon regressions
 and concentrations were adjusted by adding algae and sterile WC. Thereby,
 also the culture volume was adjusted and increased with increasing rotifer
 population size to avoid density-dependent effects and to provide
 sufficient rotifer biomass for fatty acid and carbon analyses. During the
 rotifer cultivation and the experiments, the algal diet concentration was
 kept at 3.5 mg C L<sup>-1</sup> (range 1-3.5 mg C
 L<sup>-1</sup>) and 3 mg C L<sup>-1</sup> (range
 1-3 mg C L<sup>-1</sup>), respectively. Prior to a food switch and prior
 to collecting rotifers on filters for chemical analysis, rotifers were
 sieved through a mesh (55 µm) and rinsed with sterile WC in order to
 separate them from their food. Using a stereo microscope, concentrations
 of rotifers (individuals per ml) in the rinsed cultures were
 determined. During the experiments, rotifers
 were kept at 22°C under continuous illumination (80 µmol photons
 m<sup>-2 </sup>s<sup>-1</sup>). <i>Experimental
 procedure</i> Rotifers of each strain were
 pipetted from rinsed precultures into bottles containing sterile WC and
 either <i>M. minutum</i> or <i>N. limnetica</i> as
 food (three replicates). The initial concentration of rotifer populations
 was set to 20-25 individuals per ml food suspension in an initial total
 volume of 500 ml per bottle and replicate. During the five experimental
 days, the culture volume increased steadily from initially 500 to 2200 ml
 per replicate due to the daily addition of algae and WC. After five days
 of grazing on <i>M. minutum </i>or<i> N.
 limnetica</i>, rotifers were collected on pre-combusted glass-fibre
 filters for the analysis of carbon (&gt;0.15 mg C) and fatty acids
 (&gt;0.5 mg C). Subsequently, replicates were
 combined by mixing all remaining rotifers of each treatment (either
 initially fed <i>M. minutum </i>or <i>N.
 limnetica</i>). They were split again and distributed randomly among
 three bottles, representing three replicates per treatment, filled with
 sterile WC and <i>S. elongatus</i> as food. After three days
 of grazing on <i>S. elongatus</i>, the experiments were
 terminated. Dead individuals were carefully removed from the sieved and
 rinsed rotifer cultures under a stereo microscope using a mouth pipette
 before rotifers were collected on filters for the analysis of carbon and
 fatty acids. <i>Chemical
 analysis</i> For carbon analysis, the filters
 with algal cells or rotifers were dried for at least 48 h at 50°C. POC was
 determined using an elemental analyser (Euro EA 3000, HEKAtech GmbH,
 Wegberg, Germany). For fatty acid analysis, the filters were stored at
 -20°C  and analysed following the protocol described in Wacker <i>et
 al.</i> 2016. <i>Literature</i> Michaloudi E, Papakostas S, Stamou G, Neděla V, Tihlaříková E, Zhang W, Declerck SAJ. 2018 Reverse taxonomy applied to the Brachionus calyciflorus cryptic species complex: Morphometric analysis confirms species delimitations revealed by molecular phylogenetic analysis and allows the (re) description of four species. <i>PLoS One</i> <b>13</b>, 1–25. (doi:10.1371/journal.pone.0203168) Paraskevopoulou S, Tiedemann R, Weithoff G. 2018 Differential response to heat stress among evolutionary lineages of an aquatic invertebrate species complex. <i>Biol. Lett.</i> <b>14</b>. (doi:10.1098/rsbl.2018.0498) Wacker A, Piepho M, Harwood JL, Guschina IA, Arts MT. 2016 Light-induced changes in fatty acid profiles of specific lipid classes in several freshwater phytoplankton Species. <i>Front. Plant Sci.</i> <b>7</b>. (doi:10.3389/fpls.2016.00264)