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.

Cultivation of food organisms

The phytoplankton species Synechococcus elongatus (SAG 89.79, Culture Collection of Algae, University of Göttingen, Germany), Monoraphidium minutum (SAG 243-1) and Nannochloropsis limnetica (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^-1 at 22 °C under continuous illumination of 100 µmol photons m^-2 s^-1.

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 >0.15 mg C for carbon analysis and >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). 

Cultivation of rotifers

Three strains of B. calyciflorus s.s. (‘IGB’, ‘Cornell’, ‘USA’) and one strain of the closely related species B. fernandoi (‘No.2484’) were used in the experiments (see supplementary material 1 for strain information and Michaloudi et al. (2018) and Paraskevopoulou et al. (2018) for species identification). Rotifers were precultured in bottles with sterile WC and M. minutum (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^-1 (range 1-3.5 mg C L^-1) and 3 mg C L^-1 (range 1-3 mg C L^-1), 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^-2 s^-1).

Experimental procedure

Rotifers of each strain were pipetted from rinsed precultures into bottles containing sterile WC and either M. minutum or N. limnetica 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 M. minutum or N. limnetica, rotifers were collected on pre-combusted glass-fibre filters for the analysis of carbon (>0.15 mg C) and fatty acids (>0.5 mg C).

Subsequently, replicates were combined by mixing all remaining rotifers of each treatment (either initially fed M. minutum or N. limnetica). They were split again and distributed randomly among three bottles, representing three replicates per treatment, filled with sterile WC and S. elongatus as food. After three days of grazing on S. elongatus, 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.

Chemical analysis

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 et al. 2016.

Literature

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. PLoS One 13, 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. Biol. Lett. 14. (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. Front. Plant Sci. 7. (doi:10.3389/fpls.2016.00264)