Thromboplastin

The antibiotic-supplemented diets were fed to the flies at both larval and adult stages (L+/A+), or only at the larval stage (L+/A−). For the latter, the pupae were collected and rinsed with ddH₂O twice before transferring to antibiotic-free vials. To test the maternal or paternal effects, the newly emerged males and females were crossed with their WT partners for 1–2 weeks. The F1 generation was reared on the antibiotic-free diet. Then, 100 eggs (0–6 h collection) and 10 adult females (1 day old) from each cross were collected as test samples for ELISA (Elabscience Biotechnology). In addition, 10 WT adult females (1 day old) from antibiotic-free and Tet-100-containing diets were collected as negative and positive controls, respectively. Three biological replicates were used for each cross and control. A measure of 400 µL of trichloroacetic acid solution (1%) and beads (Lysing Matrix D Bulk, Cat. 6540-434, MPbio, France) were added to the sample tube, and the tissues were homogenized using a Precellys 24 homogenizer (for 20 s at 6000 rpm). The sample tubes were centrifuged at 4,000 g for 10 min at room temperature. Then, the supernatant (27.5 µL) was mixed with reconstitution buffer (82.5 µL), and 50 µL of the mixture was used for analysis. The standard solution (1.0 ppm) was diluted according to the manufacturer’s instructions. A measure of 50 µL of diluted standard solution and sample mixture was added per well of the pre-coated 96-well microtiter plate in duplicate; 50 µL of the antibody working solution was added to each well, and the plate was covered with a lid, gently oscillated for 5 s, and incubated with shading light at 37°C for 30 min. After incubation, the microplate wells were washed six times with 250 µL/well of washing buffer, 100 μL/well of streptavidin–horseradish peroxidase (HRP conjugate) was added, and the plate was incubated at 37°C for 30 min in the dark. Then, the plate was washed again as described previously, and 50 μL of each substrate reagent A and B was added sequentially per well. After incubation at 37°C in the dark for 15 min, the reaction was stopped by adding 50 μL/well of stop solution. Optical density (OD) at 450 nm and 630 nm of each well was measured as reference wavelength (TECAN microplate reader). The concentrations (ppb) were calculated as the OD value measured at 630 nm subtracted from that measured at 450 nm. The absorbance percentage was calculated using the following formula: absorbance (%) = A/A0 × 100% (A: average absorbance of the standard solution or sample; A0: average absorbance of 0 ppb standard solution). The average absorbance value from duplicate wells was added to the standard curve. The concentration calculated from the standard curve was multiplied by 8 (the dilution factor for the pretreatment of tissue/egg samples according to the manual) for the final concentration of the samples.