Nondrop 2000

Ovary samples were removed from −80°C and placed on ice, and the entire procedure was performed on ice. RNA was extracted from the ovaries of queen bees using Trizol (Invitrogen, Carlsbad, CA, USA), and the concentration of RNA was measured using NonDrop 2000 (Thermo Scientific). The extracted RNA was then reverse transcribed using Takara kit (TaKaRa, Dalian, China). The reverse transcribed cDNA is frozen at −20°C until use.
We selected six genes [Vgr, Vg, juvenile hormone acid methyltransferase (Jhamt), Hex110, Tor, and Egfr] (Table S1) using Actin as a reference gene and performed a 10-fold dilution of the cDNA. A total of 1.6-µL forward and reverse primers, 2-µL CDNA solution, 10-µL TB Green Premix Ex Taq, and the addition of sterilized water to 20 µL. Amplification was performed using the following cycling conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 30 s, then 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. We used the 2^−ΔΔCT method and calculated the mean of three technical replicates (39 (link)). Calculation of relative differential gene expression in queen ovaries was performed after limiting queen oviposition.

The purification of lipoteichoic acid was measured by UV spectrophotometry (Nondrop 2000, Thermo, USA). First, pure water was used to zero the instrument (usually repeated three times), the sample (LTA and standard) was put into the detection arm, and then A260 nm and A280 nm wavelengths were selected for measurement. The instrument provided the value after the detection was complete [32 (link)]. By comparing with the LTA standard of Staphylococcus aureus (Sigma, USA), we were able to determine whether the extracted substance was lipoteichoic acid. Through continuous UV detection [33 (link)], the absorption peaks of nucleic acid and protein at A260 nm and A280 nm were detected, and the residues of nucleic acid and protein in the test substance was detected. This also allowed measurement of the characteristic absorption peak of lipoteichoic acid at A300 nm.

Ovary samples were removed from −80°C and placed on ice, and the entire procedure was performed on ice. RNA was extracted from the ovaries of queen bees using Trizol (Invitrogen, Carlsbad, CA, USA), and the concentration of RNA was measured using NonDrop 2000 (Thermo Scientific). The extracted RNA was then reverse transcribed using Takara kit (TaKaRa, Dalian, China). The reverse transcribed cDNA is frozen at −20°C until use.
We selected six genes [Vgr, Vg, juvenile hormone acid methyltransferase (Jhamt), Hex110, Tor, and Egfr] (Table S1) using Actin as a reference gene and performed a 10-fold dilution of the cDNA. A total of 1.6-µL forward and reverse primers, 2-µL CDNA solution, 10-µL TB Green Premix Ex Taq, and the addition of sterilized water to 20 µL. Amplification was performed using the following cycling conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 30 s, then 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. We used the 2^−ΔΔCT method and calculated the mean of three technical replicates (39 (link)). Calculation of relative differential gene expression in queen ovaries was performed after limiting queen oviposition.