Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis

Abstract

Background Molting is an important physiological process in the growth and development of arthropoda, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is a key enzyme which plays a critical role in the synthesis of ecdysone in insects, but it has not been identified in cladocera. Results The CYP302Al gene of Daphnia sinensis was cloned and its function was analyzed in this paper. The CYP302Al gene of D. sinensis was 5926 bp in full-length, with an open reading frame (ORF) of 1596 bp that encoded 531 amino acids (aa), a molecular weight of 60.82 kDa and an isoelectric point of 9.29. The amino acid sequence analysis revealed that there were five characteristic conserved regions of cytochrome P450 family (namely helix-C, helix-K, helix-I, PERF and heme-binding). In dsRNA mediated experiment, the expression level of CYP302A1 gene decreased significantly (knock-down of 56.22%) in the 5% Escherichia coli concentration treatment. In addition, the expression levels of EcR and USP and HR3 genes in the downstream were also significantly decreased, whereas that of FTZ-f1 gene increased significantly. In the 5% E. coli treatment, the molting time at maturity of D. sinensis prolonged, and the development of embryos in the incubation capsule appeared abnormal or disintegrated. The whole-mount in situ hybridization showed that the CYP302A1 gene of D. sinensis had six expression sites before RNA interference (RNAi), which located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine. However, the expression signal of the CYP302A1 gene of D. sinensis disappeared in the first antennal ganglion and obviously attenuated in the ovary after RNAi. Conclusion The CYP302A1 gene played an important role in the ecdysone synthesis pathway of D. sinensis, and the knock-down of the gene affected the molting and reproduction of D. sinensis.

The moulting arthropod: a complete genetic toolkit review

“…Genomic presence of dib (cyp302a1) as well as sad (cyp315a1) was confirmed in all the four major arthropod groups, except for Ixodes scapularis (Qu et al, 2015;Schumann et al, 2018;Miyakawa et al, 2018;Rotenberg et al, 2020;Dermauw et al, 2020;Qi et al, 2023). Similarly, shd (cyp314a1) was found in most examined arthropods, and was even cloned from Daphnia pulex and L. salmonis, except for the decapods Neocaridina denticulata, Litopenaeus vannamei, Penaeus monodon and Palaemon carinicauda (Sumiya et al, 2014;Qu et al, 2015;Sandlund et al, 2018;Ventura et al, 2018;Dermauw et al, 2020).…”

Non-Consumptive Effects of Harmonia axyridis on the Reproduction and Metabolism of Spodoptera frugiperda

“…JH regulates vitellin synthesis, oocyte maturation, and the uptake of ooxanthine by oocytes [ 41 ]. 20E, a steroid hormone synthesized and secreted by the insect’s prothoracic gland, is vital for regulating growth, development, metabolism, and reproduction [ 42 ]. In our study, we found that the non-consumptive effect of natural enemies resulted in reduced levels of JH, 20E, and vitellogenin.…”

Function Analysis of Cholesterol 7-Desaturase in Ovarian Maturation and Molting in Macrobrachium nipponense: Providing Evidence for Reproductive Molting Progress

“…Molting plays an extremely important role in the growth, development, reproduction, and survival of crustaceans and runs through their entire life cycle [ 10 ]. During the breeding period, female molting is interwoven with ovarian development, specifically manifested in the need to molt before holding eggs [ 11 ].…”