Figure Desciption.docx

<b>Figure 1.</b> Basic information on silkworm receptor tyrosine kinase <i>CAD96CA</i>. (A) Prediction of the transmembrane zone. (B) Subcellular localization. CAD96CA is predominantly located at the plasma membrane. (C) Tissue period expression profile. (D) Phylogenetic tree of different species. (E) Phylogenetic trees within species. The phylogenetic analysis was performed using the Neighbor-Joining (NJ) method in MEGA7 software.<b>Figure 2.</b> Schematic diagram of <i>CAD96CA</i> knockout vector obtained using CRISPR-Cas9. (A) gRNA transgene knockout vector. The 3×P3 promoter is a neuro-system-specific synthetic promoter sequence that drives the expression of green fluorescent protein (GFP) in silkworms, which appears as eye-specific green fluorescence during fluorescence screening (EGFP, enhanced green fluorescent protein). (B) Schematic representation of sgRNAs target sites within the gene locus. (C) Scoring and mismatch loci of gRNAs designed via the CCTOP web. (D)<i> </i><i>CAD96CA</i>-gRNA, N4-Cas9, and <i>CAD96CA</i> knockout egg-larvae-adults under green fluorescence. Under green fluorescence, only the eyes of the G1 generation have light, the N4-Cas9 body glows, and the mutant has both eyes and body. (E) Mutations at the target site were detected by polymerase chain reaction amplification. (F) Part of the mutation sequence of an individual.<b>Figure 3.</b> Mutant phenotypic observation and statistics (A) Comparison of the body size of the <i>CAD96CA</i> mutant and WT at 4LM, with WT on the left and mutant on the right. Differences among (B, C) mutant and WT silk glands, cocoons, and pupation. The left and right are the WT and mutant, respectively. (D) <i>In vitro </i>20E treatment reverts to mutant pupation failure phenotype. DMSO is on the left, and an equal volume of 20E is injected on the right. (E) Proportion of mutants that successfully pupated. (F) Individual weight difference on the 5^th instar day 0. (G) Differences in cocoon layer rate (H, I) Differential timelines of larval wandering and cuticle formation between mutations and controls. 4LM-fourth instar molt, WT-wild type, DMSO-dimethyl sulfate, 20E-20-hydroxyecdysone, 5L0-5th instar on day 0.<br><b>Figure 4.</b> Effect of <i>CAD96CA</i> genes on JH and 20E pathways (A) Transcription levels of ecdysone synthetase-related genes during the wandering stage. (B) Transcription levels of 20E-responsive genes during the wandering stage. (C) Expression levels of <i>E75B</i> at different time points. (D-E) Expression levels of genes in response to the JH pathway. (F) Expression of <i>Kr-h1</i> in fat bodies and gonads at the 4L0 and 4LM. (G) 20E and JH III determination. SW was used as an internal control primer for all qPCRs. Black bars: wild type (WT); gray bars: <i>CAD96CA</i>^mut. <i>neverland</i> (<i>NVD)</i>, <i>phantom</i> (<i>Phm)</i>,<i> shroud </i>(<i>SRO)</i>, <i>shadow</i> (<i>Sad)</i>, <i>disembodied </i>(<i>Dib)</i> and <i>Spookier </i>(<i>Spo)</i>; fourth instar on day 0 (4L0), fourth instar molt (4LM), fifth instar on day 3 (5L3). fifth instar on day 5 (5L5), eukaryotic initiation factor 4A (<i>eif4A</i>; SW). Asterisks denote significant differences between treatments and controls, as determined using the pairwise T-test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Values represent means ± SEM (N = 3).<b>Figure 5</b>. Knockout <i>CAD96CA</i> inhibits autophagy-apoptosis and MAPK signaling pathways. (A-C) Transcript levels of <i>ATG1</i>,<i> Caspase3</i>, and <i>Caspase9</i> in the fat body during the wandering stage. (D-I) Phosphorylation detection of RAF1 and ERK. Asterisks denote significant differences between treatments and controls, as determined using the pairwise T-test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001,). Values represent means ± SEM (N = 3).