Compound specific stable isotope analysis (CSIA) of amino acids (AA) extracted from flies, ontogenetic environments after fly eclosion and samples of environments prior to an experimental set-up were used to determine δ¹³C isotope values of 12 amino acids including alanine (Ala), asparagine/aspartic acid (Asx), glutamine/glutamic acid (Glx), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr) and valine (Val); asterisks denote essential amino acids. Experiments were set up in the laboratory facilities of University of Bremen, Germany in October 2021; amino acid extraction and δ¹³C measurement were executed at the University of Göttingen, Germany and the Kompetenzzentrum Stabile Isotope, Göttingen, Germany. Samples were collected to deduce fly dietary amino acid origin-bacterial, fungal or plant derived- acquired during development in relation to amino acid composition of environment after fly eclosion under five different symbiont-environment conditions. To that end, single initially axenic first-instar larvae were introduced to semi-natural conditions using 1 mL of fruit puree inoculated with 50 µL of fly fecal matter suspension collected from 30 female flies maintained in microcosms (see Riedel & Rohlfs (in preparation) for set-up of microcosms and Riedel et al. (preprint, https://doi.org/10.1101/2025.10.14.682149) for fecal matter inoculation); or 1 mL of artificial lab diet. After development to adult flies and eclosion from the pupal case, flies were collected and dried. The experimental units in which flies had developed in, were frozen along samples of fresh substrate (not inoculated with fecal matter suspension). Fly and respective samples of ontogenetic environments post eclosion were pooled using a randomized stratified approach to achieve a minimal collective fly weight of 2 g per sample. For five treatment groups five samples were pooled (nFly= 25) and the respective ontogenetic environments were pooled accordingly (nontoenv = 25). Substrate samples were dehydrated first in a drying oven then in a lyophilizer and then homogenized alongside fly samples. Amino acids were extracted as described in Larsen et al. 2016 including three external standards and nor-leucine as internal standard. Extracted amino acid mixes were then separated and analyzed using mass spectrometry following (Pollierer et al. 2020, also described in Riedel et al. (preprint, https://doi.org/10.1101/2025.10.14.682149). The triplicate measurements were aligned to the external standard and extracted measurement were inspected visually and manually corrected if necessary. Mean δ¹³C isotope values were corrected for carbon added during the derivatization of samples following O'Brien et al. (2002).

Funded by the State of Bremen