Fig 1.
Effects of HearNPV on lipid metabolism of H. armigera and effects of HFD on HearNPV-infected H. armigera larvae.
(A) LDs of healthy (CK) and HearNPV-infected (NPV) larvae at 2 dpi with Nile red. Scale bar: 25 μm. (B) LDs sizes of CK and NPV larvae. (C-D) TAG and FFA levels of CK and NPV larvae of H. armigera at 1, 2, 3, 4 and 5 days post-infection (dpi). (E) Survival of infected larvae with ND (NPV+ND) and infected larvae with HFD (NPV+HFD). (F) Relative expression level of poly in NPV+ND and NPV+HFD larvae. (G) Height at death of NPV+ND and NPV+HFD larvae. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 2.
HaAKH1 and HaAKH2 participated in lipid metabolism of HearNPV infected H. armigera larvae.
(A-B) Relative expression levels of HaAKH1 and HaAKH2 in healthy larvae (CK) and infected larvae (NPV). (C-D) Efficiency of RNAi of HaAKH1 and HaAKH2 in infected larvae treated with dsHaAKH1 and dsHaAKH2 for 24h and 48 h. (E) LDs of infected larvae treated with dsEGFP, dsHaAKH1 and dsHaAKH2 for 48 h. Scale bar: 25 μm. (F) Statistics of LDs sizes of infected larvae treated with dsEGFP, dsHaAKH1 and dsHaAKH2. (G-H) TAG and FFA levels in infected larvae of H. armigera treated with dsEGFP, dsHaAKH1 and dsHaAKH2 for 24h and 48 h. (I) LDs of infected larvae treated with PBS, mature peptides of HaAKH1 and HaAKH2 for 48 h. Scale bar: 25 μm. (J) Statistics of LDs sizes of infected larvae treated with PBS, HaAKH1 and HaAKH2. (K-L) TAG and FFA levels of infected larvae of H. armigera treated with PBS and HaAKH1 and HaAKH2 for 24h and 48 h. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 3.
HaAKH1 and HaAKH2 were involved in regulating the HearNPV infection process and behavior changes of H. armigera.
(A) Survival of infected larvae treated with dsRNAs (dsEGFP, dsHaAKH1 and dsHaAKH2). (B) Effect of dsRNAs treatments on poly mRNA (24 and 48 hours post injection) and protein (48 hours post injection) expression level. (C) Crawling distance of healthy larvae treated with dsEGFP (CK+dsEGFP), dsHaAKH1 (CK+dsHaAKH1) and dsHaAKH2 (CK+dsHaAKH2) and infected larvae treated with dsEGFP (NPV+dsEGFP), dsHaAKH1 (NPV+dsHaAKH1) and dsHaAKH2 (NPV+dsHaAKH2). (D) Height at death of infected larvae treated with dsRNAs. (E) Survival of infected larvae treated with PBS and mature peptides of HaAKH1 and HaAKH2. (F) Effect of HaAKH1 and HaAKH2 treatments on poly mRNA (24 and 48 hours post injection) and protein (48 hours post injection) expression level. (G) Crawling distance of healthy and infected larvae treated with PBS, HaAKH1 and HaAKH2. (H) Height at death of infected larvae treated with PBS, HaAKH1 and HaAKH2. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 4.
In silico and in vitro studies validated the HaAKH1–HaAKHR and HaAKH2–HaAKHR interaction.
(A) Subcellular localization of HaAKHR proteins. pEGFP meant empty vector, pEGFP—AKHR meant vector expressing HaAKHR. EGFP: EGFP fluorescent signal; Did: Did fluorescent signal, labelling cell membrane; Merge: overlapping of EGFP and Did images. Scale bar: 20 μm. (B-C) Left: Structural overview of HaAKH1-HaAKHR and HaAKH2-HaAKHR complex models. The HaAKHR protein was shown in a cartoon representation in gray, and HaAKH1 and HaAKH2 were shown in a stick representation in yellow. The active-site residues were shown in the technical representation in rainbow. Right: Zoom-in view of the predicted interface. Key interface residues in HaAKHR were shown in the technical representation and were labeled by residue name and position. Hydrogen bonds were displayed in purple and labeled with their bond lengths (Å). (D) Representative images of Ca2+ imaging after heterologous expression of HaAKHR in HEK293T cells in response to HaAKH1 and HaAKH2. pcDNA3.1—mCherry—HaAKHR meant the cells expressing the recombinant plasmid of HaAKHR with pcDNA3.1(+)—mCherry before treatment. F0 represented the initial state and FMax represented the strongest state of Ca2+ signal after treatment with PBS, HaAKH1 or HaAKH2. mCherry and Fluo-4 signal were shown in red and green, respectively; Merge: overlapping of mCherry and Fluo-4 images and shown in rainbow. Scale bar: 20 μm. (E) Fluorescence detection of Fluo-4 AM after heterologous expression of HaAKHR in HEK293T cells in response to PBS, HaAKH1 and HaAKH2. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 5.
HaAKHR involved in regulating the lipid metabolism, HearNPV infection process and behavioral changes of infected H. armigera.
(A) Relative expression level of HaAKHR in healthy (CK) and infected (NPV) larvae. (B) Efficiency of RNAi of HaAKHR in infected larvae treated with dsHaAKHR for 24h and 48 h. (C) LDs in fat bodies of infected larvae treated with dsEGFP and dsHaAKHR for 48 h. Scale bar: 25 μm. (D) Statistics of LDs sizes of infected larvae treated with dsEGFP and dsHaAKHR. (E-F) TAG and FFA levels of infected larvae of H. armigera treated with dsEGFP and dsHaAKHR for 24h and 48 h. (G) Survival of infected larvae treated with dsEGFP and dsHaAKHR. (H) Effect of dsHaAKHR on poly mRNA (24 and 48 hours post injection) and protein (48 hours post injection) expression level. (I) Crawling distance of healthy and infected larvae treated with dsEGFP and dsHaAKHR. (J) Height at death of infected larvae treated with dsEGFP and dsHaAKHR. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 6.
HaHSL was regulated by HaAKH/HaAKHR and participated in the regulation of lipid metabolism and behavioral changes in infected H. armigera larvae.
(A) Relative expression level of four triglyceride enzymes (HaHSL, HaBmm, HaLsd1 and HaLsd2) in healthy (CK) and infected (NPV) larvae at 1, 2,3 and 4 dpi. (B-C) Relative expression level of HaHSL in infected larvae of H. armigera treated with dsEGFP, dsHaAKH1, dsHaAKH2 and dsHaAKHR or treated with PBS, HaAKH1 and HaAKH2 for 24h and 48 h. (D) Relative activity level of HaHSL in CK and NPV larvae. (E-F) Relative activity level of HaHSL of infected larvae treated with dsEGFP, dsHaAKH1, dsHaAKH2 and dsHaAKHR or treated with PBS, HaAKH1 and HaAKH2 for 24 and 48 h. (G) Efficiency of RNAi of HaHSL in infected larvae treated with dsHaHSL for 24h and 48 h. (H) LDs of infected larvae treated with dsEGFP and dsHaHSL for 48 h. Scale bar: 25 μm. (I) Statistics of LDs sizes of infected larvae treated with dsEGFP and dsHaHSL. (J-K) TAG and FFA levels in infected larvae of H. armigera treated with dsEGFP and dsHaHSL for 24h and 48 h. (L) Survival of infected larvae treated with dsEGFP and dsHaHSL. (M) Crawling distance of healthy larvae treated with dsEGFP (CK+dsEGFP) and dsHaHSL (CK+dsHaHSL), and infected larvae treated with dsEGFP (NPV+dsEGFP) and dsHaHSL (NPV+dsHaHSL). (N) Height at death of infected larvae treated with dsEGFP and dsHaHSL. (Data were represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 7.
Model for HearNPV manipulating host HaAKH signaling to induce the climbing behavior.
HearNPV led to an increase in the expression of HaAKH1 and HaAKH2, and the secreted HaAKH1 and HaAKH2 peptides bound to the receptor HaAKHR located on the cell membrane of the fat body, inducing the lipase HaHSL to break down TAG in fat body, providing energy for virus replication and promoting the locomotion of infected larvae, ultimately inducing the climbing behavior. Image created with Biorender.com, with permission.