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. 2022 Sep 29:13:992306.
doi: 10.3389/fphys.2022.992306. eCollection 2022.

Cyromazine affects the ovarian germ cells of Drosophila via the ecdysone signaling pathway

Muhammad Zaryab Khalid  1 , Zhipeng Sun  1 , Jing Zhang  1 , Shijie Zhang  1 , Guohua Zhong  1
Affiliations

Cyromazine affects the ovarian germ cells of Drosophila via the ecdysone signaling pathway

Muhammad Zaryab Khalid et al. Front Physiol. .

Abstract

Cyromazine, an insect growth regulator, has been extensively used against the insect pests of livestock and households. Previously, it was observed that the continuous selection of cyromazine from the larval to the adult stage decreased the number of germline stem cells (GSCs) and cystoblasts (CBs) in the adult ovary. In addition, in this study, we observed that the number of primordial germ cells (PGCs) was also decreased in the larval ovary after treatment with cyromazine. However, the mechanism by which it affects the germ cells is yet to be explored. Consequently, to deeply investigate the effects of cyromazine on the germ cells, we performed tissue-specific RNA sequencing. Bioinformatics analysis revealed that the ecdysone signaling pathway was significantly influenced under cyromazine stress. Based on that, we screened and selected 14 ecdysone signaling responsive genes and silenced their expression in the germ cells only. Results of that showed a considerable reduction in the number of germ cells. Furthermore, we mixed exogenous 20E with the cyromazine-containing diet to rescue the ecdysone signaling. Our results supported that the application of exogenous 20E significantly rescued the germ cells in the transgenic lines. Therefore, this implies that the cyromazine decreased the number of germ cells by affecting the ecdysone signaling pathway.

Keywords: RNA interference; RNA sequencing; apoptosis; ecdysone signaling; germline stem cells; primordial stem cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Ovaries of Drosophila house different types of cells. (A) The adult ovary of Drosophila consists of 16–20 ovarioles, and the germarium of each ovariole has different types of cells. The germline stem cells (GSCs) strictly maintain and differentiate under the regulation of signals from the somatic cells. At every stage of early cell division, the GSCs are surrounded by somatic cells for their proper regulation. GSCs divide into cystoblasts (CBs), which later undergo four rounds of mitotic division to generate a 16-cells cyst. In the end, only one cell among these 16-cell cysts differentiates as an oocyte. (B) The third instar larval ovary of Drosophila, also consists of somatic cells and germ cells. Before pupation, most of the terminal filaments (TF) cells has formed. While the TF cells completely formed at the early pupal stage. The germ cells can be identified with a germ cell-specific marker (anti-vasa). However, only the germ cells with single dot-shaped spectrosome were considered as PGCs.
FIGURE 2
FIGURE 2
Effect of cyromazine on the germ cells of larval ovaries. (A) RNAi of ecdysone-responsive primary genes and the effect of cyromazine on the PGCs. (B) RNAi of Halloween genes and the effect of cyromazine on the PGCs. (C) RNAi of ecdysone-responsive genes and the effect of cyromazine on the PGCs. CK means that the flies were fed on a normal diet, while treatment means the flies were fed insecticide containing diet. (D) Larval ovary from the CK group. (E,F) Larval ovaries from the cyromazine-treated group (scale bar, 25 μm). For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
FIGURE 3
FIGURE 3
Effect of cyromazine on the GSCs of adult ovaries. (A) RNAi of ecdysone-responsive primary genes and the effect of cyromazine on the GSCs. (B) RNAi of Halloween genes and the effect of cyromazine on the GSCs. (C) RNAi of ecdysone-responsive genes and the effect of cyromazine on the GSCs. CK means that the flies were fed on a normal diet, while treatment means the flies were fed insecticide containing diet. (D) The adult ovariole from the CK group. White circle indicates GSCs, while white arrow represents CBs. (E) Only one GSCs and one CB is present in the cyromazine-treated ovariole. (F) The ovariole lacking any GSC in the germarium (scale bar, 10 μm (D,E), 25 μm (F)). For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
FIGURE 4
FIGURE 4
Effect of cyromazine on the CBs of adult ovaries. (A) RNAi of ecdysone-responsive primary genes and the effect of cyromazine on the CBs. (B) RNAi of Halloween genes and the effect of cyromazine on the CBs. (C) RNAi of ecdysone-responsive genes and the effect of cyromazine on the CBs. CK means that the flies were fed on a normal diet, while treatment means the flies were fed insecticide containing diet. (D) The adult ovariole from the CK group. White circle indicates GSCs, while white arrow represents CBs. (E) Only one GSCs and no CB can be seen against the cyromazine-treated ovariole. (F) The ovariole lacking any germ cell in the germarium (scale bar, 10 μm (D,E), 25 μm (F)). For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
FIGURE 5
FIGURE 5
Rescue experiment of PGCs. (A) The number of PGCs against ecdysone-responsive primary genes. (B) The number of PGCs against Halloween genes. (C) The numbers PGCs against ecdysone-responsive genes. The blue bars indicate the numbers of PGCs against the cyromazine treatment, while the red bars indicate the numbers of PGCs from the flies fed on a diet containing 20E with the cyromazine. For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
FIGURE 6
FIGURE 6
Rescue experiment of GSCs. (A) The number of GSCs against ecdysone-responsive primary genes. (B) The number of GSCs against Halloween genes. (C) The number of GSCs against ecdysone-responsive genes. The blue bars indicate the numbers of GSCs against the cyromazine treatment, while red bars indicate the numbers of GSCs from the flies fed on a diet containing 20E with the cyromazine. For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
FIGURE 7
FIGURE 7
Rescue experiment of CBs. (A) The number of CBs against ecdysone-responsive primary genes. (B) The number of CBs against Halloween genes. (C) The number of CBs against ecdysone-responsive genes. The blue bars indicate the number of CBs against the cyromazine treatment, while the red bars indicate the number of CBs from the flies fed on a diet containing 20E with the cyromazine. For the t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant (p > 0.05).
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