Figures and data
Effects of CLas on lipid metabolism and reproductive behavior of D. citri.
(A) Comparison of TAG levels in the fat bodies of CLas-positive (CLas+) and CLas-negative (CLas-) females 5, 9, 13 days after emergence (DAE). (B) Comparison of glycogen levels in the fat bodies of CLas+ and CLas-females 5, 9, 13 DAE. (C) Lipid droplets in fat bodies dissected from CLas+ and CLas-females 9 DAE stained with Nile red. Scale bar = 40 μm. (D-F) Comparison of the preoviposition period, oviposition period and the fecundity of CLas+ and CLas-adults. Data are shown as means ± SEM. The significant differences between CLas-positive and CLas-negative psyllids were determined using Student’s t-tests (**p < 0.01, ***p < 0.001).
DcAKH is involved in the mutualistic relationship between CLas and D. citri resulting in increased fecundity.
(A) Comparison of temporal expression patterns of DcAKH between the ovaries of CLas- and CLas+ psyllids. (B) Efficiency of RNAi of DcAKH in CLas- and CLas+ females treated with dsDcAKH for 48 h. (C) Comparison of TAG levels in fat bodies of CLas- and CLas+ females treated with dsDcAKH for 48 h. (D) Comparison of glycogen levels in fat bodies of CLas- and CLas+ females treated with dsDcAKH for 48 h. (E) Lipid droplets stained with Nile red in fat bodies dissected from CLas+ females treated with dsDcAKH for 48 h. Scale bar = 40 μm. (F) Ovary phenotypes of CLas+ females treated with dsDcAKH for 48 h. Scale bar = 200 μm. o: ovary, s: spermathecae. (G-I) Comparison of the preoviposition period, oviposition period and the fecundity between CLas- and CLas+ adults treated with dsDcAKH. (J) The CLas titer in ovaries of CLas+ females treated with dsDcAKH for 48 h. (K) Representative confocal images of CLas in the reproductive system of CLas+ females treated with dsDcAKH for 48 h. Scale bar = 200 μm. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CLas-Cy3: the CLas signal visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei and CLas. Data are shown as means ± SEM. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
DcAKHR is involved in the mutualistic relationship between CLas and D. citri resulting in increased fecundity.
(A) Comparison of temporal expression patterns of DcAKHR between the ovaries of CLas- and CLas+ psyllids. (B) The efficiency of RNAi of DcAKHR in CLas- and CLas+ psyllids treated with dsDcAKHR for 48 h. (C) Comparison of TAG levels in fat bodies of CLas- and CLas+ females treated with dsDcAKHR for 48 h. (D) Comparison of glycogen levels in fat bodies of CLas- and CLas+ females treated with dsDcAKHR for 48 h. (E) Lipid droplets stained with Nile red in fat bodies dissected from CLas-positive females treated with dsDcAKHR for 48 h. Scale bar = 40 μm. (F) Ovary phenotypes of CLas+ females treated with dsDcAKHR for 48 h. Scale bar = 200 μm. o: ovary, s: spermathecae. (G-I) Comparison of the preoviposition period, oviposition period and the fecundity of CLas- and CLas+ adults treated with dsDcAKHR. (J) The CLas titer in the ovaries of CLas+ females treated with dsDcAKHR for 48 h. (K) Representative confocal images of the reproductive system of CLas+ females treated with dsDcAKHR for 48 h. Scale bar = 200 μm. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CLas-Cy3: the CLas signal visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei and CLas. Data are shown as means ± SEM. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, ***p < 0.001).
Identification and validation of the target relationship between miR-34 and DcAKHR.
(A) The putative binding sites of miRNAs in the DcAKHR 3’-UTR as predicted by miRanda and RNAhybrid. (B) Dual-luciferase reporter assays using HEK293T cells co-transfected with miRNA agomir and recombinant pmirGLO vectors containing the predicted binding sites for miR-2, miR-14 and miR-34 in the CDS of DcAKHR. (C) Dual-luciferase reporter assays using HEK293T cells co-transfected with miR-34 agomir plus recombinant pmirGLO vectors containing DcAKHR-3’UTR or mutated DcAKHR-3’UTR. (D) Tissue expression pattern of miR-34 in CLas+ female adults at 7 DAE in the head, ovary, fat body, and midgut. (E) Comparison of temporal expression patterns of miR-34 in ovaries of CLas- and CLas+ females. (F) Effect of miR-34 agomir and antagomir treatments on DcAKHR mRNA expression and protein level in ovaries of CLas- and CLas+ psyllids after 48 h. (G) Relative expression of miR-34 targeted DcAKHR in vivo as demonstrated by an RNA immunoprecipitation assay. Data are shown as mean ± SEM. For B-D, significant differences among the different treatments are indicated by lowercase letters above the bars (one-way ANOVA followed by Tukey’s Honestly Significant Difference test at P < 0.05). The significant differences between treatment and control are indicated by asterisks in E-G (Student’s t-test, **p < 0.01, ***p < 0.001).
miR-34 participation in mutualistic interactions between D. citri and CLas.
(A) Comparison of TAG levels in fat bodies of CLas- and CLas+ females treated with agomir-34 for 48 h. (B) Comparison of glycogen levels in the fat bodies of CLas- and CLas+ females treated with agomir-34 for 48 h. (C) Lipid droplets stained with Nile red in fat bodies dissected from CLas+ females treated with agomir-34 for 48 h. Scale bar = 40 μm. (D) Ovary phenotypes of CLas+ female treated with agomir-34 for 48 h. Scale bar = 200 μm. o: ovary, s: spermathecae. (E-G) Comparison of the preoviposition period, oviposition period and the fecundity between CLas- and CLas+ adults treated with agomir-34. (H) CLas titer in ovaries of CLas+ females treated with agomir-34 for 48 h. (I) Representative confocal images of CLas in the reproductive system of CLas+ females treated with agomir-34 for 48 h. Scale bar = 200 μm. The signals of DAPI and CLas-Cy3 are same as described in Figure 2. Data are shown as means ± SEM. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, ***p < 0.001).
The JH signaling pathway is regulated by AKH signaling pathway and is involved in the increase in fecundity of D. citri induced by CLas.
(A) JH titer in the abdomen of CLas+ females treated with dsDcAKH for 48 h. (B) Effects of dsDcAKH treatment on mRNA level of JH signaling pathway in fat bodies of CLas+ females. (C) Effects of dsDcAKH treatment on mRNA levels of components of the JH signaling pathway in the ovaries of CLas+ females. (D) JH titers in the abdomens of Clas+ females treated with dsDcAKHR for 48 h. (E) Effects of dsDcAKHR treatment on mRNA level of JH signaling pathway in fat bodies of CLas+ females. (F) Effects of dsDcAKHR treatment on mRNA levels of components of the JH signaling pathway in ovaries of CLas+ females. (G) JH titer in abdomen of CLas+ females treated with agomir-34 for 48 h. (H) Effects of agomir-34 treatment on mRNA levels of components of the JH signaling pathway in fat bodies of CLas+ females. (I) Effects of agomir-34 treatment on mRNA levels of components of the JH signaling pathway in the ovaries of CLas+ females.
Mechanisms linking metabolism and reproduction of D. citri induced by CLas.
After infection with CLas, the TAG and glycogen levels in fat bodies of CLas-positive psyllids significantly increased as well as the size of lipid droplets. In ovaries, CLas upregulates the AKH/AKHR signaling and downregulates miR-34 to increase lipid metabolism and activate JH-dependent vitellogenesis, thereby improving the fecundity of CLas-positive females. D. citri are more fecund than their uninfected counterparts. The interaction between CLas and D. citri affecting reproduction is a win-win strategy; the more offspring of D. citri, the more CLas in the field.
Sequence characterization of AKH from different insect species (data are related to Figure 2).
(A) Alignment of the amino acid sequences of the DcAKH transmembrane domain with homologs from other insect species. The sequences in the green box represent the mature peptides (QVNFSPNW). (B) Phylogenetic analysis of DcAKH and its homologs in other insect species. The phylogenetic tree was constructed using neighbor-joining with 1000 bootstrap replicates; values > 50% are shown on the tree. The scale bar indicates the number of amino acid substitutions per site.
Tissue expression of DcAKH in CLas-positive adult females 9 days after emergence in the head, ovary, fat body, and midgut (data are related to Figure 2).
In vivo and in vitro studies validating the DcAKH-DcAKHR interaction (data are related to Figure 3).
The predicted tertiary structure of the DcAKHR protein. The seven conserved transmembrane ion channels are shown as TM1-TM7. (B) Phylogenetic analysis the protein sequences of AKHR. The phylogenetic tree was constructed using neighbor-joining with 1000 bootstrap replicates; values > 50% are shown on the tree. The scale bar indicates the number of amino acid substitutions per site. (C) Concentration-response curves for the effect of Ca2+ on DcAKHR-expression in CHO cells. (D) The expression levels of DcAKHR in CLas-negative and CLas-positive females treated with dsDcAKH for 48 h.
Tissue expression of DcAKHR in CLas-positive female adults 9 DAE in the head, ovary, fat body, and midgut (the data are related to Figure 3).
The expression levels of miR-34 in CLas-negative and CLas-positive females treated with agomir-34 for 48 h (the data are related to Figure 4).
(A-E) Melting curve for qRT-PCR primers of DcAKH, DcAKHR, Dcβ-ACT, miR-34 and U6.
