Phase-related differences in egg production of the migratory locust regulated by differential oosorption through microRNA-34 targeting activinβ

Abstract

Outbreaks of locust plagues result from the long-term accumulation of high-density egg production. The migratory locust, Locusta migratoria, displays dramatic differences in the egg-laid number with dependence on population density, while solitarious locusts lay more eggs compared to gregarious ones. However, the regulatory mechanism for the egg-laid number difference is unclear. Herein, we confirm that oosorption plays a crucial role in the regulation of egg number through the comparison of physiological and molecular biological profiles in gregarious and solitarious locusts. We find that gregarious oocytes display a 15% higher oosorption ratio than solitarious ones. Activinβ (Actβ) is the most highly upregulated gene in the gregarious terminal oocyte (GTO) compared to solitarious terminal oocyte (STO). Meanwhile, Actβ increases sharply from the normal oocyte (N) to resorption body 1 (RB1) stage during oosorption. The knockdown of Actβ significantly reduces the oosorption ratio by 13% in gregarious locusts, resulting in an increase in the egg-laid number. Based on bioinformatic prediction and experimental verification, microRNA-34 with three isoforms can target Actβ. The microRNAs display higher expression levels in STO than those in GTO and contrasting expression patterns of Actβ from the N to RB1 transition. Overexpression of each miR-34 isoform leads to decreased Actβ levels and significantly reduces the oosorption ratio in gregarious locusts. In contrast, inhibition of the miR-34 isoforms results in increased Actβ levels and eventually elevates the oosorption ratio of solitarious locusts. Our study reports an undescribed mechanism of oosorption through miRNA targeting of a TGFβ ligand and provides new insights into the mechanism of density-dependent reproductive adaption in insects.

Fig 1. Oosorption differences between gregarious and solitarious locusts.

(A) Normal and resorbed terminal oocytes at different stages. N, normal terminal oocyte; RB, resorption body. Scale bar, 2 mm. (B) Ovariole number of gregarious or solitarious adults at PAE0, 2, 4, 6, 8, 10 and 12. n = 15. (C) Oosorption ratio of gregarious or solitarious adults at PAE0, 2, 4, 6, 8, 10 and 12. n = 10–18. (D) The number of resorbed terminal oocytes and eggs in the first egg pod in the gregarious or solitarious adults. n = 15–16. **P < 0.01, ***P < 0.001.

Fig 2. Transcriptomic analyses of gregarious (G) and solitarious (S) terminal oocytes (TO).

(A) The number of differentially expressed genes between GTO and STO. (B and C) qRT-PCR verification of the top 5 genes upregulated in GTO (B) or STO (C). n = 7–12. (D) The expression levels of nine verified genes at N and RB1 stages. n = 4–7. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. Effects of Actβ knockdown on oosorption.

(A) Neighbor joining tree construction of TGFβ ligand subfamily in four species. Consensus unrooted trees were generated with 1000 bootstrap trials using the neighbor-joining method. Lm, Locusta migratoria; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens. (B) Gene expression level of Actβ in the terminal oocytes after dsGFP or dsActβ injection. n = 9–10. (C) Protein expression level of Actβ in the terminal oocytes after dsGFP or dsActβ injection. n = 4. (D) Oosorption ratio after dsGFP or dsActβ injection. n = 18. (E) Length of terminal oocytes after dsGFP or dsActβ injection. n = 8–9. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4. Identification of miRNAs targeting Actβ.

(A) Diagram of miRNA target sites on the Actβ mRNA sequence. Three DNA fragments with target sites were inserted into psiCHECK-2 vectors for dual-luciferase reporter assays. (B) Dual-luciferase reporter assays using S2 cells cotransfected with miRNA mimics and recombinant psiCHECK2 vectors containing the predicted binding sites of respective miRNAs. miR-NC, negative control. n = 5–6. (C) The binding sites (in red) of Actβ were mutated into bases that were noncomplementary to the miR-34 seed region (in blue). WT, predicted binding sites; MT, mutated binding sites. (D) Dual-luciferase reporter assays after site mutation. n = 6. (E) Colocalization of miR-34a and Actβ by FISH in the follicle cells of terminal oocytes. Green, Actβ; red, miR-34a. Arrows indicate Actβ/miR-34a colocalized areas. Scale bar: 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (F) The relative miRNA abundance of miR-34b or miR-34c in the terminal oocytes after agomir-NC or agomir-34a injection. n = 8–9. (G) The relative abundance of precipitated Actβ mRNA by Ago1-RIP in the terminal oocytes after agomir-NC or agomir-34a injection. n = 4.

Fig 5

The expression levels of three miR-34 isoforms in the GTO and STO (A) or in the N and RB1 (B). (A) n = 12–15. (B) n = 6–8. *P < 0.05.

Fig 6. Effects of agomir-34 injection on oosorption in the gregarious locusts.

(A) The relative miRNA abundance of miR-34a, miR-34b or miR-34c in the terminal oocytes after injection with respective agomirs. n = 7–8. (B) Gene expression level of Actβ in the terminal oocytes after injection with agomir of miR-34a, miR-34b or miR-34c. n = 5–8. (C) Protein expression level of Actβ in the terminal oocytes after injection with agomir of miR-34a, miR-34b or miR-34c. n = 4. (D) Oosorption ratios after injection with agomir of miR-34a, miR-34b or miR-34c. n = 10–13. (E) Length of terminal oocytes after injection with agomir of miR-34a, miR-34b or miR-34c. n = 8–13. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 7. Effects of antagomir-34 injection on oosorption in the solitarious locusts.

(A) The relative miRNA abundance of miR-34a, miR-34b or miR-34c in the terminal oocytes after injection with respective antagomirs. n = 7–8. (B) Gene expression level of Actβ in the terminal oocytes after injection with antagomir of miR-34a, miR-34b or miR-34c. n = 8–17. (C) Protein expression level of Actβ in the terminal oocytes after injection with antagomir of miR-34a, miR-34b or miR-34c. n = 4. (D) Oosorption ratios after injection with antagomir of miR-34a, miR-34b or miR-34c. n = 17–25. (E) Length of terminal oocytes after injection with antagomir of miR-34a, miR-34b or miR-34c. n = 9–17. *P < 0.05, **P < 0.01, ***P < 0.001.