The receptor tyrosine kinase torso regulates ecdysone homeostasis to control developmental timing in Bombyx mori

Abstract

Insect growth and development are precisely controlled by hormone homeostasis. The prothoracicotropic hormone (PTTH) receptor, Torso, is a member of the receptor tyrosine kinase family in insects. Activation of Torso by PTTH triggers biosynthesis and release of the steroid hormone in the prothoracic gland (PG). Although numbers of genes functioning in steroid hormone synthesis and metabolism have been identified in insects, the PTTH transduction pathway via its receptor Torso is poorly understood. In the current study, we describe a loss-of-function analysis of Torso in the silkworm, Bombyx mori, by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori Torso (BmTorso) did not eventually affect larval ecdysis and metamorphosis processes. Instead, BmTorso deficiency resulted in significant extension of developing time during larval and pupal stages with increased pupa and cocoon sizes. The ecdysteriod titers in the hemolymph of BmTorso mutants sharpy declined. Transcriptional levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in BmTorso-deficient animals. Additionally, RNA-Seq analysis revealed that genes involved in the longevity pathway and protein processing in the endoplasmic reticulum pathway were affected after BmTorso deletion. These results indicate that Torso is critical for maintaining steroid hormone homeostasis in insects.

Keywords: Bombyx mori; CRISPR/Cas9; Torso; developmental timing.

Fig. 1

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA‐guided Cas9 nucleases) system mediated disruption of BmTorso. (A) Schematic diagram of the BmTorso locus structure and single guide (sgRNA)‐targeted sites. Two sgRNA targeting sites, TS1 and TS2, are located at the anti‐sense strand of exon 4 and the sense strand of exon 5, respectively. The sequences of sgRNA‐targeted sites are in green. The red letters show the protospacer adjacent motif (PAM) sequences. (B) Sequence of mutations at TS1 and TS2 induced by CRISPR/Cas9. The dashes in every sequence line represent deleted residues and the detailed indel size is shown on the right. The base of point mutation is shown in blue.

Fig. 2

Developmental delay and increased body size in △Torso animals. (A) The stages of larval and pupal development in WT (wild‐type) and △Torso (L1, 1st instar; L2, 2nd instar; L3, 3rd instar; L4, 4th instar; L5, 5th instar; P, pupae). (B) The upper graph shows male pupae and cocoons for the indicated genotype. The down graph showed female pupae and cocoons for the indicated genotype. (C) The cocoon shell weight of WT and △Torso. (D) The cocoon shell ratio of WT and △Torso. The data are shown as the mean ± SEM (n = 30). ***P < 0.001 according to two‐tailed t‐test.

Fig. 3

The ecdysteroid titers and expression differences of ecdysone pathway genes in △Torso animals. (A) Relative ecdysteriod titers in the hemolymph at wandering stage of wild‐type (WT) and △Torso. Hemolymph was collected from 10 larvae, and the pooled sample was used to determine ecdysteriod titers. (B) Hemolymph ecdysteroid titers of WT and △Torso during pupal stage. (C). Expression change of Halloween genes in prothoracic gland (PG) at wandering stage. (D) Expression change of ecdysone responsive genes in fat body (FB) at wandering stage. The data are shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 according to two‐tailed t‐test.

Fig. 4

RNA‐Seq analysis reveals the differentially expressed genes (DEGs) between wild‐type (WT) and △Torso. (A) Gene Ontology functional classification of the DEGs. (B) The top five enriched Kyoto Encyclopedia of Genes and Genomes pathways. (C) Quantitative real‐time polymerase chain reaction validation for the genes associated with the regulation of longevity and protein processing signaling pathway. The data are shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 according to two‐tailed t‐test.