System, Integumentary

VSc was performed on a weekly basis throughout the entire laying period (20th–71st week of life) containing 51 study days, on which the plumage and integument condition of more than 20,000 hens were assessed. Each week, a sample of 200 hens per genetic strain [27 ] was scored in five previously defined locations per compartment (n = 20 hens per location). The locations were chosen specifically for this barn ([13 (link)], modified) and included sections of the littered scratching areas, the intermediate ceiling of the aviary, the slats in front of the nest boxes, and the perches, respectively. For visual assessment, the hens’ body was divided into five regions: head/neck, back, tail, wing, and breast/belly ([26 (link)], modified). All of the body regions were scored for plumage and integument damage using a five and four point scale, respectively (Table 1). Concerning plumage condition, feather loss was ranked according to its degree, with no difference being made between the types of the respective feathers (flight feathers, other contour feathers, semiplumes). Integument damage was defined as fresh or crusted lesions affecting the skin only or also deeper tissues. Scars were not considered. Any other visible abnormalities, for instance lesions at the hens’ vent or toes, were recorded without a fixed scoring scheme.
In addition, HSc was carried out on seven study days (n = 60 hens per genetic strain and day) using the same scoring scheme as for the visual method. The dates for HSc (21st, 30th, 40th, 47th, 56th, 65th, and 70th week of life) were chosen according to a pilot study [21 ], in which particularly risk-bearing periods for the occurrence of feather pecking and cannibalism were identified. Both VSc and HSc were performed by the same observer on all of the study days. In the weeks in which both VSc and HSc were carried out, the observer started with the visual assessment. Once the entire sample was scored visually, HSc was conducted visiting each compartment of the aviary in a randomly different order to that in the VSc method.

The laboratory stock colony of L. serricorne, originally collected in 2014 from a tobacco warehouse in Guizhou Province, China, was reared on Chinese medicinal material (Angelica sinensis) and maintained at 28 ± 1 °C and 40% ± 5% relative humidity under a scotoperiod of 24 h.
Samples at different developmental stages, including early larvae (EL, <24 h post-hatching), late larvae (LL, older than fourth instar larvae and before prepupae), pupae (PU, >48 h post-pupation), early adults (EA, <24 h post-eclosion), and late adults (LA, one week old) were collected separately and stored at −80 °C. In the tissue-specific experiment, the fifth instar larvae were used for tissue isolation. The integument, fat body, gut, and carcass of L. serricorne were dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). Each tissue type was placed in a 1.5-mL centrifuge tube containing RNA storage reagent (Tiangen, Beijing, China). Pools of 30 individuals of larvae were used to prepare the integument, gut, and carcass, and 50 individuals were pooled to collect the fat body tissue. All tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C. Each sample was replicated three times.

A previously reported method was used to determine the chitin content (Arakane et al., 2005 (link)) of larvae collected and weighed at 3, 6, 12, 24, 48, 72, and 96 h after the injection of siRNA. Each analysis was performed using three replicates of 15 larvae. The larvae were dissected in normal saline to obtain the midgut and integument samples, which were placed in a 1.5-mL centrifuge tube and then ground to a powder in liquid nitrogen. After adding 500 µl 6% KOH solution, the ground samples were heated at 80°C for 90 min and then centrifuged at 12,000 g for 20 min at 4°C. The supernatant was discarded and each sample was suspended in 1 mL PBS buffer and then centrifuged at 12,000 g for 20 min at 4°C. After discarding the supernatant, each sample was resuspended in 200 µl Mcllvaine buffer (pH 6.0) (Tianzd, Beijing, China). To hydrolyze chitin to N-acetyl glucosamine (GlcNAc), 50 µl chitinase from Streptomyces griseus (Sigma-Aldrich, Shanghai, China) was added to individual samples, which were then incubated for 72 h at 37°C.
The GlcNAc concentrations were determined using a modified Morgan-Elson assay (Reissig et al., 1955 (link)). Briefly, various GlcNAc concentrations (0.00025, 0.0005, 0.001, 0.002, 0.004, and 0.008 mol/L) were used as standards. The samples incubated for 72 h were centrifuged at 12,000 g for 20 min at 4°C. For each sample, 10 µl supernatant and different GlcNAc standards were added to new centrifuge tubes. Next, 10 µL 0.27 mol/L sodium tetraborate was added to individual samples, which were then heated at 99.9°C for 10 min. The samples were immediately cooled to room temperature and then mixed with 100 µl 10% DMAB solution (10 g p-dimethylaminobenzaldehyde in a solution comprising 12.5 ml concentrated hydrochloric acid and 87.5 ml glacial acetic acid, diluted 1:10 with glacial acetic acid). The samples were heated at 37°C for 20 min and then centrifuged at 12,000 g for 5 min at 4°C. An 80-µl aliquot of each sample was transferred to a 96-well plate, and the absorbance at 585 nm was recorded. Standard curves were prepared using the different GlcNAc concentrations.

Total RNA was extracted from insects at different developmental stages (i.e., first-day first to sixth instar larvae, prepupae, pupae, and adults) and from various tissues (i.e., foregut, midgut, hindgut, fat body, salivary gland, Malpighian tubules, and integument). The MsTPS expression levels in different M. separata developmental stages and tissues were analyzed by quantitative real-time PCR (qRT-PCR), with Msβ-actin (GenBank accession number: GQ856238) and MsGAPDH (glyceraldehyde-3-phosphate dehydrogenase; GenBank accession number: HM055756) used as reference genes. Primer Premier 5.0 was used to design qRT-PCR primer pairs MsTPS-q-F and MsTPS-q-R, Msβ-actin-q-F and Msβ-actin-q-R, and MsGAPDH-q-F and MsGAPDH-q-R (Supplementary Table S1). The qRT-PCR mixture comprised 2 µl cDNA, 6.8 µl ddH₂O, 0.6 µl sense and anti-sense primers, and 10 µl THUNDERBIRD SYBR qPCR Mix kit (Toyobo, Shanghai, China). The qRT-PCR was performed using the SimpliAmp PCR instrument (Thermo Fisher Scientific, MA, United States of America), with the following PCR conditions: 95°C for 3 min; 40 cycles of 94°C for 10 s and 57°C for 30 s. The data were recorded using the Bio-Rad CFX Manager 3.1 software. Melting curves were checked to assess the specificity of the qRT-PCR. Moreover, primer efficiency was validated before analyzing gene expression. The qRT-PCR was completed using three technical replicates and three biological replicates.