Grace insect medium

The wild-type PRV virulent SD-2017 strain was isolated in 2017 from a pig farm in Shandong, China and was preserved in the Chinese General Microbiological Culture Collection Center (No. 22,047). The SD-2017 gE gene (Acc. No. MW535262) was used to identify the PRV variant strain circulating in China. In order to avoid the mutation of parent strains caused by passing culture, we used SD-2017 strain of the lower generation (F10) in this study. This strain had limited adaptability on PK15 cells and had no significant difference in virus titer and plaque size compared with Bartha-K/61. The PRV Bartha-K61 vaccine strain was obtained from Shandong Huahong Biological Engineering Co., LTD, and was stored in our lab. Bartha-K/61 strain has a deletion of 3,489 bp nucleotides at positions 122,560 − 126,049, including part of the gI gene, all the gE and Us9 genes, and part of the Us2 gene. PK15 porcine kidney cells were cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) at 37 °C in 5% CO₂ in a humidified incubator. Protein purification utilized Sf9 cells (ATCC CRL-1711) to propagate recombinant baculovirus and were cultured in Grace insect medium (Gibco) containing 2% FBS at 27 °C.

The larvae were first washed with distilled water and then immersed briefly in 70% (v/v) ethanol to sterilize their surfaces and reduce contamination. Larval hemolymph was then collected from incisions in the last proleg. Freely-dripping hemolymph was collected into sterile polypropylene 1.5 ml centrifuge tubes preloaded with Grace Insect Medium (GIM; Gibco) with added gentamycin (10 mg/ml; Gibco), amphotericin B (250 μg/ml; Gibco) and 0.1 mM phenylthiourea (PTU; Sigma-Aldrich). Fresh hemolymph (8–10 drops; one drop 26 μl contains about 1.3 x 10⁵ of cells) was mixed with 300 μl of GIM. The hemolymph suspension was immediately transferred to μ-slide VI cell culture plates (IBIDI). Each plate hole was filled with 100 μl of hemolymph mixed with GIM. Following this, the plates were incubated for four or 24 hours, depending on the group, at 27°C and 80–90% humidity.

DaZao, a bivoltine strain of the silkworm, was obtained from the Silkworm Gene Bank of Southwest University, Chongqing, China. The non-diapause eggs were divided into two groups; one group was incubated at 25 °C (diapause induction temperature), while the other group was incubated at 15 °C (non-diapause induction temperature). All the eggs were incubated at the same light condition (continuous darkness). Four groups were prepared from embryos exposed to the two temperature treatments (25 and 15 °C) and in the two phases (the blastokinesis (BK) and head pigmentation (HP) phases). Due to the non-uniform development of embryos, we dissected a great deal of the silkworm eggs and selected the embryos showing the same morphology at the same developmental stage for subsequent sequencing. The resulting four groups were labeled BK25, BK15, HP25, and HP15. Each group comprised 100 embryos. All samples were stored at −80 °C for further use. The cell line, BmE, derived from the silkworm embryo, was cultured at 27 °C in Grace insect medium (Life Technologies, Shanghai, China) supplemented with 10% fetal bovine serum (United States Biological, Swampscott, MA, USA), penicillin (200 U/mL), and streptomycin (200 U/mL).

Pre-chilled fifth instar larvae were sterilized with 70% ethanol. The fat body was then dissected in sterile PBS and transferred to a 6-well cell culture plate containing 2 ml Grace insect medium (Life Technologies, USA) and different concentrations (2, 5 and 10 µg ml⁻¹) of JH (Sigma-Aldrich, USA) or solvent (acetone). Samples were incubated at 27°C for 8 h followed by total RNA isolation.

Midgut cells were harvested using a modified protocol [61 (link)] from animals that were in the last two instars of their developmental period. We followed the published protocol for insect cells [62 ] to maintain the primary cell culture. Briefly, midguts were placed in insect physiological solution (NaCl 178 mM, KCl 4.3 mM, CaCl₂ 4.3 mM, NaHCO₃ 3.8 mM, 0.5% gentamicin, 0.01% antibiotic antimycotic PH 6.5) and washed twice before transferring to a well on a 6-well plate. Cells were maintained in Grace Insect Medium (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% heat-inactivated fetal bovine, 0.1% gentamicin, vitamin mixture, and 0.1% antibiotic antimycotic at 25 °C. After 24 h, primary cell culture was filtered using 70 µm cell strainers (CLS431751, Thermo Fisher Scientific, Rockford, IL, USA) to remove gut explants. Cells were collected after gentle pipetting and washed twice in 0.1 M cold PBS buffer (8.00 g NaCl, 0.20 g KCl, 1.29 g Na₂HPO₄·3H₂O, 0.20 g KH₂PO₄, 1000 mL ddH₂O, pH 7.4) to be used in further analysis.