2.4.2. Life History Parameters

We photographed all the individuals collected in each variant from the lateral side under a dissecting microscope connected with the camera and computer. The length and height of each *Daphnia* were measured in the photographs using the NIS program (Nikon Nis Elements). The length was measured from the top of the eye to the base of the tail spine and the height was measured along the body, starting from its greatest dimension. Based on these measurements, the body volume of each individual was calculated, assuming an ellipsoidal shape for each individual according to the formula 4/3*π* × 1/2*a* × 1/2*b* × 1/2*c*, where *a* stands for the length, *b* the height, and *c* the width (assuming that the body width is equal to the body height [59]. For each ovigerous female, we counted the eggs, and the volume of an egg (upon the mean dimensions for at least two eggs in the clutch) was calculated using the same formula as that which was employed for body volume evaluation. Finally, the clutch volume as the common currency of the reproductive investment was calculated as the number of eggs multiplied by the mean egg volume for each ovigerous female.

#### 2.4.3. Metabolomic Diversity of Gut Microbiota

The extracted *Daphnia* guts were homogenised in 1 mL of milliQ water and transferred into 15 mL of their respective media, which were earlier filtered through 0.45 μm Nylon filters and autoclaved. The standard method of a Biolog EcoPlate [60,61] was used to analyse the metabolic diversity of the gut microbiota in these samples. An EcoPlate (Biolog, Hayward, CA, USA) was employed to measure the ability of the bacterial community to utilise the different carbon substrates. An EcoPlate is a 96-well microplate composed of the triplicates of control wells (containing no additional carbon source) and 31 wells containing various carbon sources (Table 1).

**Table 1.** Different carbon sources used by the Biolog EcoPlate method to measure the ability of the gut bacterial community of *Daphnia* to utilise carbon substrates.


Twelve plates were used, one for each variant. Each well of a single plate, except the control one (filled with milliQ water), was filled with 150 μL aliquots of homogenised and the diluted content of *Daphnia* guts from one variant. We incubated the plates in darkness at a temperature of 22 ◦C for 72 h. Because of the reduction in the tetrazolium chloride by the electrons which derived mainly from the oxidation chains, the colour in the wells increased the proportionally to the respiration rate. We measured the absorbance every four hours at 590 nm wavelength using a Biotek Synergy H1 plate reader (Biotek Corporation, Broadview, IL, USA). For the analysis, we used the maximal colour development rates (Vmax). In calculating the Vmax values, we used Gen 5 software (Biotec Corporation, Broadview, IL, USA). Additionally, we identified the slope for every four consecutive reads. The Vmax was calculated using a linear regression model by determining the maximum slope during the 72 h incubation time. For the maximal rate of colour development, the Vmax (mOD × min−1) was treated as an indicator of the intensity of the respiration of a single carbon source by the *Daphnia* gut microbial communities. The difference in the Vmax between the carbon-containing wells and the control wells was calculated to determine the influence of each additional carbon source on the respiration rate (delta). When the delta was lower than zero or equal to zero, we assumed the microorganism community did not use the source, and we set this value of Vmax to zero. For the analyses of the communitylevel physiological fingerprinting, we used the relative values of the respiration rate for each carbon source, calculated as the percentage share of each carbon source in the sum of the Vmax for the whole plate. The overall microbial activity in each microplate was expressed as the mean Vmax for the plate, and was calculated as the average of all the wells, including the control.
