The Function of LmPrx6 in Diapause Regulation in Locusta migratoria Through the Insulin Signaling Pathway

Abstract

Peroxiredoxins (Prxs), which scavenge reactive oxygen species (ROS), are cysteine-dependent peroxide reductases that group into six structurally discernable classes: AhpC-Prx1, BCP-PrxQ, Prx5, Prx6, Tpx, and AhpE. A previous study showed that forkhead box protein O (FOXO) in the insulin signaling pathway (ISP) plays a vital role in regulating locust diapause by phosphorylation, which can be promoted by the high level of ROS. Furthermore, the analysis of transcriptome between diapause and non-diapause phenotypes showed that one of the Prxs, LmPrx6, which belongs to the Prx6 class, was involved. We presumed that LmPrx6 might play a critical role in diapause induction of Locusta migratoria and LmPrx6 may therefore provide a useful target of control methods based on RNA interference (RNAi). To verify our hypothesis, LmPrx6 was initially cloned from L. migratoria to make dsLmPrx6 and four important targets were tested, including protein-tyrosine phosphorylase 1B (LmPTP1B), insulin receptor (LmIR), RAC serine/threonine-protein kinase (LmAKT), and LmFOXO in ISP. When LmPrx6 was knocked down, the diapause rate was significantly reduced. The phosphorylation level of LmPTP1B significantly decreased while the phosphorylation levels of LmIR, LmAKT, and LmFOXO were significantly increased. Moreover, we identified the effect on two categories of genes downstream of LmFOXO, including stress tolerance and storage of energy reserves. Results showed that the mRNA levels of catalase and Mn superoxide dismutase (Mn-SOD), which enhanced stress tolerance, were significantly downregulated after silencing of LmPrx6. The mRNA levels of glycogen synthase and phosphoenolpyruvate carboxy kinase (PEPCK) that influence energy storage were also downregulated after knocking down of LmPrx6. The silencing of LmPrx6 indicates that this regulatory protein may probably be an ideal target for RNAi-based diapause control of L. migratoria.

Keywords: ISP; LmPrx6; RNA interference; diapause rate.

Figure 1

The hypothetical molecular relationships of LmPrx6 (Prx6 of Locust migratoria) in ISP in an adult female: Female adults sense the short photoperiod (SP) and cause enhanced LmPrx6 that inhibits ROS. The reduction of ROS leads to the phosphorylation of PTP1B. The phosphorylated LmPTP1B inhibits IR, which could phosphorylate AKT. The attenuation of AKT leads to the dephosphorylation of FOXO. Then, the dephosphorylated FOXO transfers to the nucleus to induce the expression of genes of stress tolerance (catalase and Mn-SOD) and genes of storage of energy reserves (phosphoenolpyruvate carboxy kinase (PEPCK) and glycogen synthase).

Figure 2

(A): Amplification of LmPrx6 (672 bp). M: DL 2000 DNA Maker; 1: LmPrx6; (B): Synthesis of dsLmPrx6 (T7+LmPrx6, 710 bp). M: DL2000 DNA Maker; 1: dsLmPrx6.

Figure 3

(A): Multiple sequence alignment with 15 sequences of PRX6 from different species; (B): the predicted 3-D structure of LmPrx6 with 28.18% α-helix, 19.55% extended strand, 5% β-turn, and 47.27% random coil; (C): phylogenetic analysis with 14 sequences of PRX6 from different species. The tree was generated with MEGA6 after CLUSTAL W alignment using the amino acid sequence of Aedes aegypti (AAG47823.1), Sepiella maindroni (AEI52300.1), Gryllotalpa orientalis (AAX18657.1), Drosophila melanogaster (AAG47823.1), Tribolium castaneum (XP_968419.1), Hymeniacidon perlevis (ABB91779.1), Scylla paramamosain (ACJ53746.1), Rattus norvegicus (NP_446028.1), Homo sapiens (NP_004896.1), Penaeus monodon (AQW41375.1), Oplegnathus fasciatus (ADJ21808.1), Crassostrea gigas (CAK22382.1), Anopheles gambiae (XP_320690.3), Bombyx mori (NP_001040386.1), Laodelphax striatellus (RZF48233.1), Onthophagus Taurus (XP_022899820.1), Diuraphis noxia (XP_015373818.1), Halyomorpha halys (XP_014281612.1), Locust migraytoria (MT563098), Locust migratoria (MT890637), and Prx3 of Drosophila busckii (ALC46298.1) was used as outgroup.

Figure 4

(A), Relative mRNA level of LmPrx6 in the whole body of adult females under an SP (short photoperiod) and LP (long photoperiod) with and without dsLmPrx6 injection. (B), The comparison of the expression of LmPrx6 with and without dsLmPrx6 injection under SP and LP. (C), Diapause rate detected after injecting dsLmPrx6 under LP. (D), Diapause rate detected after injecting dsLmPrx6 under SP. All results were expressed as means ± standard error (SE) of the three replicates. * Indicates probability level of p < 0.05 by Student’s t-test.

Figure 5

(A), Phosphorylation level of LmPTP1B, LmIR, LmAKT, and LmFOXO under both long and short photoperiods (LP, SP). (B), Phosphorylation level of LmPTP1B, LmIR, LmAKT, and LmFOXO in the control compared to dsLmPrx6 treatment under both LP and SP. All results were expressed as means ± standard error (SE) of the three replicates. (C), Relative mRNA level with and without dsLmPrx6 injection of FOXO, catalase, glycogen synthase, Mn-SOD, and PEPCK under LP compared to SP. (D), Relative mRNA level of FOXO, catalase, glycogen synthase, Mn-SOD, and PEPCK in CK compared to dsLmPrx6 treatment under both LP and SP. * Indicates probability level of p < 0.05 by Student’s t-test.

Figure 6

(A), ROS activity in the whole body of adult females under long and short photoperiods. (B), ROS activity detected in the whole body of adult females after injecting dsLmPrx6 under SP and LP. All results were expressed as means ± standard error (SE) of the three replicates.* Indicates probability level of p < 0.05 by Student’s t-test.

Figure 7

(A), Catalase, Mn-SOD, glycogen synthase, and PEPCK activities in the whole body of adult females under long and short photoperiods. (B), The comparison of catalase, Mn-SOD, glycogen synthase, and PEPCK activities detected in the whole body of adult females with and without injection of dsLmPrx6 under SP and LP. All results were expressed as means ± standard error (SE) of three replicates. * Indicates probability level of p < 0.05 by Student’s t-test.