Functional analysis of zona pellucida domain protein Dusky in Tribolium castaneum

Abstract

The zona pellucida domain protein Dusky (Dy) plays a vital role in wing morphogenesis in insects, but little information on its function has been reported. In this study, we found that dy regulated wing cell size, larval and pupal duration, and the metabolism of amino acid and 20‐hydroxyecdysone in Tribolium castaneum. Using RNA‐seq, 413 differentially expressed genes were identified between physiological buffer‐injected and dy‐double‐stranded RNA‐treated larvae, including 88 downregulated genes and 325 upregulated genes. Among these genes, dy knockdown increased CYP18A1 expression to elevate the 26‐hydroxylation of 20‐hydroxyecdysone, which ultimately led to growth defects in wing cells. Silencing of dy upregulated the transcription of genes encoding tyrosine aminotransferase, 4‐hydroxyphenylpyruvate dioxygenase, homogentisate 1, 2‐dioxygenase, and Pale to promote the catabolism of tyrosine and phenylalanine, which eventually reduced amino acid content. Furthermore, dy knockdown upregulated 4E‐BP expression, and 4E‐BP silencing partially phenocopied dy RNA interference‐mediated wing morphogenesis. These results suggest that Dy controls 20‐hydroxyecdysone and amino acid metabolism to regulate wing morphogenesis in the insect.

The zona pellucida protein piopio regulates the metamorphosis and reproduction in Tribolium castaneum

“…We found that RNAi-mediated knockdown of pio inhibited the pupation and eclosion in T. castaneum (Figures 4 and 5). As previously reported, the ZP proteins dusky, dusky-like and miniature were shown to have crucial roles in metamorphosis of T. castaneum (Li et al, 2017(Li et al, , 2016(Li et al, , 2022, supporting the role of Pio in insect metamorphosis. Insect metamorphosis is coordinately controlled by 20E and JH in opposite manners (Jindra et al, 2013;Yamanaka et al, 2013).…”

“…On the third, fourth, and fifth days after injection, 10 larvae were collected from the ds‐ pio and ds‐ ver groups, respectively. Half of the samples were prepared as previously to detect 20E titer using an LTQ XL mass spectrometer system (Thermo Fisher Scientific Inc.) (Hikiba et al, 2013; Li et al, 2022). The other half of the samples were dissolved in a 1:1 mixture of methanol and ether (Sinopharm Chemical Reagent Co., Ltd.), and then homogenized.…”

“…These studies mainly reported their activities in microstructure such as embryonic denticles, wing hairs, extracellular matrix and microtubule, and so on. We have found that dusky, dusky‐like, and miniature played important roles in metamorphosis of Tribolium castaneum (Li et al, 2016, 2017, 2022), and so it is likely that other ZP proteins have unknown functions during insect development.…”

Identification and characterization of CYP307A1 as a molecular target for controlling the small hive beetle, Aethina tumida

“…In addition, AtCYP18A1 was highly expressed in the wings of Aethina tumida , which had not been reported in other insects. However, in T. castaneum , the zona pellucida domain protein Dusky (Dy) was found to modulate 20E metabolism through CYP18A1 expression, to control wing morphogenesis 43 . Thus, we speculate that CYP18A1 may play an important role in the wing development of insects.…”

Arpc2 integrates ecdysone and juvenile hormone metabolism to influence metamorphosis and reproduction in Tribolium castaneum

“…After centrifugation, the supernatants were dried and suspended with 200 μL methanol to measure 20-hydroxyecdysone (20E) titer using an LTQ XL mass spectrometer system (Thermo Fisher Scientific, Inc., San Jose, CA, USA). 35,36 The remaining samples were homogenized by dissolving a 1:1 mixture of methanol and ether. After adding n-hexane, shook the samples at 4 °C overnight, and then centrifuged them to acquire the supernatants.…”