Fig 1.
Parasitization by Cotesia vestalis influences systematic lipid contents and intestinal lipid droplet accumulation in Plutella xylostella larvae.
(A) Schematic diagram of the procedures for lipid synthesis, transportation, storage and utilization, modified from Kamareddine et al [88]. Tachykinins (TKs) in midgut enteroendocrine cells (EEs) could suppress lipogenesis in intestinal enterocytes (ECs) via TK receptors. (B) Levels of triglycerides (TGs) in C. vestalis-parasitized and nonparasitized host larvae among different developmental stages (n = 10 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01; ***: p < 0.001). 3L: Late 3rd instar; 4E: Early 4th instar; 4M: Middle 4th instar; 4L: Late 4th instar. (C) Relative levels of TG in hemolymph from C. vestalis-parasitized and nonparasitized host larvae among different developmental stages (n = 30 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01; ***: p < 0.001). (D) Fluorescent images of the middle regions of midguts from 3L, 4E, 4M and 4L C. vestalis-parasitized and nonparasitized P. xylostella larvae. Lipids were stained with BODIPY (green), and nuclei were labeled with DAPI (blue). Scale bars: 100μm.
Fig 2.
Plutella xylostella TK is highly expressed in the midgut.
(A) Relative mRNA levels of PxTK in P. xylostella larvae among different developmental stages (n = 5 for each group). Values represent the means ± SD of three independent experiments. 3M: Middle 3rd instar; 3L: Late 3rd instar; 4E: Early 4th instar; 4M: Middle 4th instar; 4L: Late 4th instar. (B) Relative mRNA levels of PxTK in eight different tissues of 4L P. xylostella larvae (n = 30 per tissue). Values represent the means ± SD of three independent experiments. (C) Upper panel: Schematic diagram of the P. xylostella larval gut; Lower panel: Localization of TK in the midgut (anti-TK, red). TK was localized in the cytoplasm in EEs (white arrowheads). ECs were labeled by BODIPY (green, yellow arrowheads), and nuclei were labeled by DAPI (blue). Scale bars: 50μm.
Fig 3.
PxTK/PxTKR signaling plays critical roles in maintaining lipid metabolism.
(A) Dose-response curves of HEK293 cells stably expressing PxTKR and treated with PxTKs. All data were taken from at least three independent experiments. Values represent the means ± SD of three independent experiments. The 50% effective concentration (EC50) for each PxTK peptide is shown on the right. (B) Relative mRNA levels of TK in P. xylostella larvae at 3 days post PxTK silencing with dsGFP treatment as a control (n = 5 for each group). Three biological replicates were performed. Data are the means ± SD; significance was determined by Student’s t-test (***: p < 0.001). (C) Relative mRNA levels of TKR in P. xylostella larvae at 3 days post PxTKR silencing with dsGFP treatment as a control (n = 5 for each group). Three biological replicates were performed. Data are the means ± SD; significance was determined by Student’s t-test (***: p < 0.001). (D) Relative levels of triglycerides (TGs) in midguts from dsPxTK-, dsPxTKR- and dsGFP (CK)-treated P. xylostella larvae at 3 days post microinjection (n = 30 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of five independent experiments (***: p < 0.001). (E) Relative levels of TG from the whole body of dsPxTK-, dsPxTKR- and dsGFP (CK)-treated P. xylostella larvae at 3 days post microinjection (n = 10 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of three independent experiments (*: p < 0.05). (F) Relative levels of TG from the whole body of P. xylostella larvae at 3 days post microinjection of chemically synthesized individual PxTK peptides (TK1, TK2, TK3, TK4, TK5 and TK6) and their mixture (n = 5 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of three independent experiments (**: p < 0.01).
Fig 4.
Cotesia vestalis parasitization upregulates host intestinal TK expression.
(A) Relative mRNA levels of PxTK in C. vestalis-parasitized and nonparasitized host larvae among different developmental stages (n = 5 for each group). Data were analyzed by Tukey’s-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). 3L: Late 3rd instar; 4E: Early 4th instar; 4M: Middle 4th instar; 4L: Late 4th instar. (B) Relative mRNA levels of PxTK in midguts from C. vestalis-parasitized and nonparasitized host larvae at different developmental stages (n = 30 for each group). Data were analyzed by Tukey’s-test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01). (C) Immunostaining for TK (red) in the middle region of midguts from C. vestalis-parasitized and nonparasitized 3L P. xylostella larvae. Nuclei were labeled by DAPI (blue). Scale bars: 50μm. (D) The number of TK-labeled cells in the middle region of midguts from C. vestalis-parasitized and nonparasitized 3L P. xylostella larvae (n = 8). Data were analyzed by Student’s t-test. Values represent the means ± SD of more than three independent experiments (*: p < 0.05).
Fig 5.
Elevation of host lipids influences the development of wasp offspring and the subsequent parasitic ability.
(A) Relative mRNA levels of PxTK in parasitized host larvae at 3 days post PxTK silencing with dsGFP treatment as a control (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). (B) Relative mRNA levels of PxTKR in parasitized host larvae at 3 days post PxTKR silencing with dsGFP treatment as a control (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). (C) Relative levels of triglycerides (TGs) in nonparasitized, C. vestalis-parasitized, C. vestalis-parasitized plus dsPxTK-treated (P+dsTK), C. vestalis-parasitized plus dsPxTKR-treated (P+dsTKR) and C. vestalis-parasitized plus dsGFP-treated (P+dsGFP) P. xylostella larvae at 3 days post microinjection (n = 10 for each group). Data were analyzed by Tukey’s-test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01; ns: not significant). (D) The pupation rate of C. vestalis in dsPxTK-treated (blue curve, n = 210), dsPxTKR-treated (purple curve, n = 169), dsGFP-treated (red curve, n = 206) and nontreated (black curve, n = 248) P. xylostella larvae. (E) The wasp emergence rate of C. vestalis in dsPxTK-treated (n = 210), dsPxTKR-treated (n = 169), dsGFP-treated (n = 206) and nontreated (n = 248) P. xylostella larvae. Data were analyzed by 2X2 chi-square-test (*: p < 0.05; ns: not significant). (F) The male: female ratios of C. vestalis that emerged from dsPxTK-treated (n = 147), dsPxTKR-treated (n = 113), dsGFP-treated (n = 162) and nontreated (n = 199) P. xylostella larvae. Data were analyzed by the 2X2 chi-square test (*: p < 0.05; **: p < 0.01; ns: not significant). (G) The parasitism rates of female C. vestalis that emerged from dsPxTK-treated (n = 242), dsPxTKR-treated (n = 242), dsGFP-treated (n = 233) and nontreated (n = 250) P. xylostella larvae. Data were analyzed by the 2X2 chi-square test (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant).
Fig 6.
Symbiotic bracovirus affects host lipid levels.
(A) Levels of triglycerides (TGs) in C. vestalis-pseudoparasitized and nonparasitized host larvae at different developmental stages (n = 10 for each group). Data were analyzed by Tukey’s test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01). 3L: Late 3rd instar; 4E: Early 4th instar; 4M: Middle 4th instar; 4L: Late 4th instar. (B) Relative mRNA levels of PxTK in C. vestalis-pseudoparasitized and nonparasitized host larvae at different developmental stages (n = 5 for each group). Data were analyzed by Tukey’s-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). (C) Levels of TG in 3L P. xylostella larvae after CvBV injection at a dose of 0.05 FE (female equivalents) per host larva (n = 10 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). (D) Fluorescent images of midguts from P. xylostella larvae with or without CvBV injection. Lipids were stained with BODIPY (green), and nuclei were labeled with DAPI (blue). Scale bars: 100 μm. (E) Relative mRNA levels of PxTK in whole larvae and midguts from P. xylostella larvae with or without CvBV injection (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (*: p < 0.05; ***: p < 0.001).
Fig 7.
A bracovirus gene, CvBV 9–2, is responsible for host lipid level reduction.
(A) Relative mRNA levels of CvBV 9–2 in parasitized P. xylostella larvae at 1 day post CvBV 9–2 silencing with dsGFP treatment as a control (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (*: p < 0.05). (B) Relative mRNA levels of CvBV 9–5 in parasitized P. xylostella larvae at 1 day post CvBV 9–5 silencing with dsGFP treatment as a control (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (**: p < 0.01). (C) Relative mRNA levels of CvBV 22–6 in parasitized P. xylostella larvae at 1 day post CvBV 22–6 silencing with dsGFP treatment as a control (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (***: p < 0.001). (D) Relative levels of triglycerides (TGs) in nonparasitized, C. vestalis-parasitized, C. vestalis-parasitized plus dsCvBV 9-2-treated (P+dsCvBV9-2), C. vestalis-parasitized plus dsCvBV 9-5-treated (P+dsCvBV9-5) and C. vestalis-parasitized plus dsGFP-treated (P+dsGFP) P. xylostella larvae 1 day post injection (n = 15 for each group). Data were analyzed by Tukey’s-test. Values represent the means ± SD of three independent experiments (*: p < 0.05; **: p < 0.01; ns: not significant). (E) Relative levels of TG in P. xylostella larvae 1 day post injection with BacCvBV 9–2 and BacGFP (control) (n = 15 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (*: p < 0.05). (F) Fluorescent images of midguts from P. xylostella larvae 1 day post injection with BacCvBV 9–2 and BacGFP. Lipids were stained with BODIPY (green), and nuclei were labeled with DAPI (blue). Scale bars: 100μm. (G) Relative mRNA levels of PxTK in the whole body and midgut of P. xylostella larvae 1 day post injection with BacCvBV 9–2 and BacGFP, respectively (n = 5 for each group). Data were analyzed by Student’s t-test. Values represent the means ± SD of three independent experiments (**: p < 0.01).
Fig 8.
Model for manipulation of host tachykinin signaling and lipid metabolism by a C. vestalis bracovirus gene, CvBV 9–2.
Schematic diagram of a model for the processes by which a C. vestalis bracovirus gene, CvBV 9–2, reduces the lipid level of P. xylostella larvae by upregulating intestinal TK expression. During parasitism, C. vestalis bracovirus is injected into P. xylostella larvae along with the eggs and then infects the different host tissues. A bracovirus gene, CvBV 9–2, is responsible for the induction of PxTK in enteroendocrine cells of the host midgut. Thus, the increase in PxTK restrains lipid production in enterocyte cells through the PxTK receptor. The reduction in host lipids during parasitism is important for the development of C. vestalis larvae, female wasp survival and subsequent parasitic efficiency.