Figure 1.
The dopamine pathway may be targeted by miR-133.
(A) miR-133 target sites were predicted in the pale and henna genes of Locusta migratoria (lmi), Drosophila species (dme), Anopheles gambiae (aga), Apis mellifera (ame), and Nasonia vitripennis (nvi). (B) The expression levels of miR-133 were determined in the brains of fourth instar gregarious (G) and solitary (S) locust nymphs using qPCR. (C, D) The expression levels of henna and pale were determined in the brains of fourth instar gregarious and solitary locust nymphs using qPCR and western blot analyses. (E) The expression levels of miR-133 were determined in the brains of fourth instar gregarious nymphs after isolation (IG) and in solitary nymphs after crowding (CS) using qPCR. (F–H) The expression levels of henna and pale were determined in the brains of fourth instar nymphs over the course of IG and CS using qPCR and western blot analyses. The qPCR data are presented as the mean ± SEM (n = 6). The western blot bands were quantified using densitometry and are expressed as the mean ± SEM (n = 4). *p<0.05; **p<0.01.
Figure 2.
miR-133 targets the coding region of henna, but it targets the 3′ UTR of pale.
(A, B) The interactions between miR-133 and the target binding sites of henna (A) and pale (B) in migratory locusts were determined using luciferase assays. (C) The strategy used to generate the plasmid expression vectors HC1, HC2, HC1M, PC1, PC2, and PC2M. (D) The mRNA expression levels of henna were determined in S2 cells co-transfected with the plasmid expression vectors HC1, HC2, HC1M, and agomir-133 using qPCR. (E) The mRNA expression levels of pale were determined in S2 cells co-transfected with the plasmid expression vectors PC1, PC2, PC2M, and agomir-133 using qPCR. (F) The protein expression levels of henna were determined in S2 cells co-transfected with the plasmid expression vectors HC1, HC2, HC1M, and agomir-133 by western blot analysis. (G) The protein expression levels of pale were determined in S2 cells co-transfected with the plasmid expression vectors PC1, PC2, PC2M, and agomir-133 by western blot analysis. The results were normalized to the expression of β-actin. Co-transfection with the empty PAC-5.1/V5 His A vector or the agomir-control (agomir-NC) was used as a negative control. HC1: CDS of henna; HC2: both the CDS and 3′UTR of henna; HC1M: HC1 containing four seed element mutations in the coding region of henna. PC1: CDS of pale; PC2: both the CDS and 3′ UTR of pale; PC2M: PC2 containing four seed element mutations in the 3′ UTR of pale. The data for the luciferase activities and qPCR analyses are presented as the mean ± SEM (n = 6). The western blot bands were quantified using densitometry and are expressed as the mean ± SEM (n = 4). *p<0.05; **p<0.01.
Figure 3.
miR-133 can interact with henna/pale in the locust protocerebrum.
(A) The combined in situ analyses of miRNA-133 and henna/pale by the co-labeling of miRNA FISH and immunohistochemistry for miRNA target were conducted to determine the co-localization between these molecules in the locust brain. The squares specifically indicate the areas where miR-133, henna, and pale were localized in the locust protocerebrum. Where green (henna and pale) and red signals (miR-133) overlap, a yellow signal is seen, indicating the co-localization of miR-133 and its targets. The images were visualized using an LSM 710 confocal fluorescence microscope (Zeiss) at a magnification of 10× (the small squares) and 63× (the large squares), respectively. (B–D) RIP was performed with an anti-Ago-1 antibody; normal mouse IgG was used as a negative control. RT-PCR (B) or qPCR (C, D) analysis was performed to amplify the henna and pale mRNA from the Ago-1 immunoprecipitates from extracts of protocerebrum tissue treated with the miR-133 agomir (agomir-133) compared to the agomir-controls (agomir-NC). M, DNA marker. The data for the RIP assay are presented as the mean ± SEM (n = 6). **p<0.01.
Figure 4.
miR-133 controls dopamine production by regulating henna and pale expression in the locust brain.
(A, B) The expression levels of miR-133 were determined 48 h after injection in gregarious locusts (G) and in solitary locusts (S) after treatment with agomir or antagomir, respectively (14 or 42 pmol) using qPCR. (C, D) The effects of 42 pmol of agomir- and antagomir-133 treatment 48 h after injection on the mRNA expression levels of henna and pale in gregarious and solitary locust brains were studied using qPCR. (E, F) The effects of 42 pmol of agomir- and antagomir-133 48 h after injection on the protein expression levels of henna and pale in gregarious and solitary locust brains were studied using western blot analysis. (G) Dopamine production after treatment with 42 pmol agomir or antagomir-133 treatment in gregarious and solitary locusts brains was evaluated using HPLC-MS. The qPCR and HPLC-MS data are shown as the mean ± SEM (n = 6). The western blot bands were quantified using densitometry and are expressed as the mean ± SEM (n = 4). *p<0.05; **p<0.01.
Figure 5.
miR-133 fosters the phase transition phenotype of the migratory locust.
(A) The effects of 14 or 42 pmol agomir-133 treatment on the behavior of gregarious locusts were studied 48 h after injection. (B) The effects of 14 or 42 pmol antagomir-133 treatment on the behavior of solitary locusts were studied 48 h after injection. Pgreg, probabilistic metric of gregariousness. The vertical lines indicate the median Pgreg values. Pgreg = 1 indicates fully gregarious behavior, and Pgreg = 0 indicates fully solitary behavior.
Figure 6.
The dopamine pathway is the direct effector for mediating the miR-133-regulated behavioral transition.
(A) The behavioral rescue experiment in gregarious locusts was performed by increasing the dopamine content through injection with the dopamine receptor agonist (DRA), R-(-)-apomorphine, or a saline control in locusts pre-treated with agomir-133. (B, C) The behavioral rescue experiment in solitary locusts was performed by injecting dsRNA against henna/pale into the locusts pre-treated with antagomir-133. Pgreg, probabilistic metric of gregariousness. The vertical lines indicate the median Pgreg values. Pgreg = 1 indicates fully gregarious behavior, and Pgreg = 0 indicates fully solitary behavior.