Dusky-like is required for epidermal pigmentation and metamorphosis in Tribolium castaneum

Abstract

Dusky-like (Dyl) is associated with the morphogenesis of embryonic denticle, adult sensory bristle and wing hair in Drosophila melanogaster. And whether Dyl involved in insect post-embryonic development and its signal transduction are poorly understood. Here, phylogenetic analysis revealed that dyl displayed one-to-one orthologous relationship among insects. In Tribolium castaneum, dyl is abundantly expressed at the late embryonic stage. Tissue-specific expression analysis at the late adult stage illustrated high expression of dyl in the fat body and ovary. Knockdown of dyl resulted in the defects in larval epidermal pigmentation and completely blocked the transitions from larval to pupal and pupal to adult stages of T. castaneum. We further discovered that dyl RNAi phenotypes were phenocopied by blimp-1 or shavenbaby (svb) silencing, and dyl was positively regulated by blimp-1 through svb in T. castaneum. These results suggest that Dyl functions downstream of Blimp-1 through Svb for larval epidermal pigmentation and metamorphosis. Moreover, ftz-f1 was down-regulated after RNA interference (RNAi) suppressing any of those three genes, indicating that Ftz-f1 works downstream of Dyl to mediate the effects of Blimp-1, Svb and Dyl on metamorphosis in T. castaneum. This study provides valuable insights into functions and signaling pathway of insect Dyl.

Figure 1. Phylogenetic tree of insect dy and dyl by Neighbor-joining method.

TC NO. indicates the T. castaneum gene; GB NO. indicates the A. mellifera gene; PHUM NO. indicates the P. h. humanus gene; ACYPI NO. indicates the A. pisum gene; BGIBMGA NO. and XP_004923535 indicate B. mori genes; AGAP NO. indicates A. gambiae gene; CG NO. indicates the D. melanogaster gene. The tree is rooted by D. melanogaster no mechanoreceptor potential A (nompA) (CG13207). The bootstrap value below 60% was removed from phylogenetic tree.

Figure 2

Developmental (a) and tissue (b) expression patterns of dyl in T. castaneum by qRT-PCR. EE, early eggs; LE, late eggs; EL, early larvae; LL, last-instar larvae; EP, early pupae; LP, late pupae; EA, early adults; LA, late adults. Tissues were isolated from adults (10-day old). One μg of total RNA from each sample was converted into cDNA for qRT-PCR.

Figure 3

Knockdown of dyl affected T. castaneum pupation, eclosion (a) and reduced ftz-f1 level (b). IB, beetles injected with physiological buffer; ds-dyl, beetles injected with dyl dsRNA. Three individuals of each group were used to extract total RNA for qRT-PCR analysis.

Figure 4

Svb RNAi affected pupation and eclosion (a) and reduced the expressions of dyl, miniature, singed, forked (b) and ftz-f1 (c) in T. castaneum. IB, beetles injected with physiological buffer; ds-svb, beetles injected with svb dsRNA. Three larvae or pupae of each group were used to extract total RNA for qRT-PCR analysis.

Figure 5

Blimp-1 RNAi affected pupation and eclosion (a) and down-regulated the mRNA levels of svb and its targeted genes (b) and ftz-f1 (c) in T. castaneum. IB, beetles injected with physiological buffer; ds-blimp-1, beetles injected with blimp-1 dsRNA. Three larvae or pupae of each group were used to extract total RNA for qRT-PCR analysis.

Figure 6. Preliminary regulation model of Dyl in T. castaneum.