Computational protocol: The Effect of Diet Mixing on a Nonselective Herbivore

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Protocol publication

[…] Six ten litre bottles each with eight litres of an algal growth medium [] were inoculated with similar biovolumes of six green algal species (all from the Culture Collection of Algae at the University of Cologne, CCAC,, ). All cultures were grown in an environmental chamber at 20°C with a 150 μmol photons s-1 m-2 light (PAR) intensity under continuous aeration. After 1 month, the batch cultures were harvested by centrifugation and the resulting pellets were freeze-dried. The animals were originally collected in a pond in Appeldorn, NRW Germany, with permission of the owner of the land. The experiment did not involve endangered or protected species. All conditions for animal maintenance and experiments were carefully optimized to meet the animals’ requirements based on extensive prior experience []. A specific ethical approval by the university’s IACUC is not required for work with gastropods according to German law. Nevertheless, we undertook all necessary measures to minimize any animal suffering and adhered to the guidelines for the use of animal behaviour for research and teaching (Animal Behaviour 83:301–309). Eggs from adult individuals of the freshwater gastropod L. stagnalis, were hatched and reared in aquaria filled with aerated tap water. The snails were fed ad libitum with (Tetra PlecoMin™) fish food pellets (Tetra, Melle, Germany). The shell height (from the apex to the lower edge of the aperture) was determined to the nearest 0.02 mm using a calliper. A cohort of 64 two-week old L. stagnalis with a shell height of 2.2 ± 0.3 mm were selected for the experiment. Of these, eight had their shells removed under a dissecting microscope and their soft bodies were dried at 60°C for three days and then weighed with a microbalance (Mettler UTM2, Giessen, Germany) to the nearest microgram to determine the initial dry mass. The remaining 56 snails were subdivided into seven treatments each containing eight replicates. The experiment was conducted in a climatized chamber at 20 ± 0.5°C. The snails were individually placed into square polyethylene containers (length = 11 cm) with 100 ml aged and aerated tap water each. The seven treatments consisted of snails fed with a mixture of all six algae species or one of the six single algal species in saturating and equal quantities.The snails were transferred into new containers every other day and water and food were renewed on a daily basis. The shell height of the snails was measured in three days intervals. During the course of the experiment, the amount of food provided was gradually increased from 1.5 to 26 mg per individual and day to avoid growth limitation by food quantitybased upon previously estimated ingestion rates (unpublished data). The algae were mixed and then transferred to the snails’ containers through hollow glass cylinders (d = 2.3 cm, h = 2.5 cm). The glass cylinders were placed in the centre of the containers covered half in water. The algae were then added through the cylinder. After 30 min when the algae had sunk to the bottom of the container the cylinder was carefully removed. This was done in order to avoid the algae from dispersing inside the containers and thereby enabling the snails to selectively feed. After 45 days, the experiment was terminated and the dry mass of the snails was determined as described above.Juvenile growth increment in L. stagnalis is assumed to be exponential []. Hence, to determine the somatic growth rate [d-1] of L. stagnalis, the following equation was used: g=ln(mend)−ln(mstart)days[d] where the mstart is the mean dry mass of the eight snails desiccated at the beginning of the experiment and mend is the dry mass of the snail individual from the respective experimental unit at the end of the experiment (day 45). The relationship between shell length and dry mass of the snails was tested in SigmaPlot (v.11, SysStat) via a nonlinear regression for exponential growth (single, 2 parameter). A one-way ANOVA followed by Tukey’s HSD was conducted in Statistica version 10 to test for significant differences between the somatic growth rates of the snails under the various food regimes. The different food treatments were set as independent variable and the somatic growth rates of the snails were set as dependent variable. Prior to the statistical tests, all data were checked for homoscedasticity using Levene’s test in Statistica. A Mann-Whitney U test was conducted in Statistica to test for a significant difference between the mean somatic growth rates of all single algae treatments and the mixed algal treatment, as the data were not homoscedastic. In order to test for differences in shell heights over time, the data was log transformed and a repeated-measures ANOVA was conducted, followed by post-hoc comparisons with Tukey’s HSD in Statistica. Some of the snails did not survive the experiment which is why the number of replicates varied between 6–8 among the treatments. […]

Pipeline specifications

Software tools SigmaPlot, Statistica
Application Miscellaneous