Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by ‘Candidatus Liberibacter asiaticus’
- National Key Laboratory of Green Pesticide, Department of Entomology, College of Plant Protection, South China Agricultural University, China
- School of Science, Western Sydney University, Australia
- Henry Fok School of Biology and Agriculture, Shaoguan University, China
Figures
Figure 1
Effects of Candidatus Liberibacter asiaticus (CLas) on lipid metabolism and reproductive behavior of D. citri.
(A) Comparison of triacylglycerides (TAG) levels in the fat bodies of CLas-positive (CLas+) and CLas-negative (CLas-) females 5, 9, and 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. In A and B, data are shown as means ± SEM with nine biological replications of at least 30 nymphs for each replication. For D-F, at least 30 female adults for each group. The significant differences between CLas-positive and CLas-negative psyllids were determined using Student’s t-tests (**p<0.01, ***p<0.001).
Figure 2 with 3 supplements
DcAKH is involved in the mutualistic relationship between Candidatus Liberibacter asiaticus (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 hr. (C) Comparison of triacylglycerides (TAG) levels in fat bodies of CLas- and CLas+ females treated with dsDcAKH for 48 hr. (D) Comparison of glycogen levels in fat bodies of CLas- and CLas+ females treated with dsDcAKH for 48 hr. (E) Lipid droplets stained with Nile red in fat bodies dissected from CLas+ females treated with dsDcAKH for 48 hr. Scale bar = 40 μm. (F) Ovary phenotypes of CLas+ females treated with dsDcAKH for 48 hr. 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 hr. (K) Representative confocal images of CLas in the reproductive system of CLas+ females treated with dsDcAKH for 48 hr. Scale bar = 200 μm. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CLas-Cy3: the CLas signal is visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei and CLas. Data are shown as means ± SEM with at least nine independent biological replications. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, *p<0.05, **p<0.01, ***p<0.001).
Figure 2—figure supplement 1
Sequence characterization of adipokinetic hormone (AKH) from different insect species.
(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.
Figure 2—figure supplement 2
Tissue expression of DcAKH in Candidatus Liberibacter asiaticus (CLas)-positive adult females 9 days after emergence in the head, ovary, fat body, and midgut.
Data are shown as means ± SEM with at least nine independent biological replications.
Figure 2—figure supplement 3
Melting curve for qRT-PCR primers of DcAKH, DcAKHR, Dcβ-ACT, miR-34, and U6.
Figure 3 with 2 supplements
DcAKHR is involved in the mutualistic relationship between Candidatus Liberibacter asiaticus (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. The protein molecular weight for DcAKHR is 48 KDa and for Dcβ-ACT is 42 KDa. (C) Comparison of triacylglycerides (TAG) levels in fat bodies of CLas- and CLas+ females treated with dsDcAKHR for 48 hr. (D) Comparison of glycogen levels in fat bodies of CLas- and CLas+ females treated with dsDcAKHR for 48 hr. (E) Lipid droplets stained with Nile red in fat bodies dissected from CLas-positive females treated with dsDcAKHR for 48 hr. Scale bar = 40 μm. (F) Ovary phenotypes of CLas+ females treated with dsDcAKHR for 48 hr. 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 hr. (K) Representative confocal images of the reproductive system of CLas+ females treated with dsDcAKHR for 48 hr. Scale bar = 200 μm. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CLas-Cy3: the CLas signal is visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei and CLas. Data are shown as means ± SEM with at least nine independent biological replications. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, ***p<0.001).
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Figure 3—source data 1
Original file for the Western blot analysis in Figure 3A and B (anti-DcAKHR and anti-β-ACT).
- https://cdn.elifesciences.org/articles/93450/elife-93450-fig3-data1-v1.zip
Figure 3—figure supplement 1
In vivo and in vitro studies validating the DcAKH-DcAKHR interaction.
The predicted tertiary structure of the DcAKHR protein. The seven conserved transmembrane ion channels are shown as TM1-TM7. (B) Phylogenetic analysis of 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 Chinese hamster ovary (CHO) cells. (D) The expression levels of DcAKHR in Candidatus Liberibacter asiaticus (CLas)-negative and CLas-positive females treated with dsDcAKH for 48 hr.
Figure 3—figure supplement 2
Tissue expression of DcAKHR in Candidatus Liberibacter asiaticus (CLas)-positive female adults 9 days after emergence (DAE) in the head, ovary, fat body, and midgut.
Figure 4 with 1 supplement
Identification and validation of the target relationship between miR-34 and DcAKHR.
(A) The putative binding sites of miRNAs in the DcAKHR 3’-untranslated region (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 Candidatus Liberibacter asiaticus (CLas) + female adults at 7 days after emergence (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) Effects of miR-34 agomir and antagomir treatments on DcAKHR mRNA expression and protein level in ovaries of CLas- and CLas + psyllids after 48 hr. The protein molecular weight for DcAKHR is 48 KDa and for Dcβ-ACT is 42 KDa. (G) Relative expression of miR-34 targeted DcAKHR in vivo as demonstrated by an RNA immunoprecipitation assay. Data are shown as mean ± SEM with nine independent biological replications. 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).
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Figure 4—source data 1
Original file for the Western blot analysis in Figure 4F (anti-DcAKHR and anti-β-ACT).
- https://cdn.elifesciences.org/articles/93450/elife-93450-fig4-data1-v1.zip
Figure 4—figure supplement 1
The expression levels of miR-34 in Candidatus Liberibacter asiaticus (CLas)-negative and CLas-positive females treated with agomir-34 for 48 hr.
Figure 5
miR-34 participation in mutualistic interactions between D. citri and Candidatus Liberibacter asiaticus (CLas).
(A) Comparison of triacylglycerides (TAG) levels in fat bodies of CLas- and CLas + females treated with agomir-34 for 48 hr. (B) Comparison of glycogen levels in the fat bodies of CLas- and CLas + females treated with agomir-34 for 48 hr. (C) Lipid droplets stained with Nile red in fat bodies dissected from CLas + females treated with agomir-34 for 48 hr. Scale bar = 40 μm. (D) Ovary phenotypes of CLas + female treated with agomir-34 for 48 hr. 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 hr. (I) Representative confocal images of CLas in the reproductive system of CLas +females treated with agomir-34 for 48 hr. Scale bar = 200 μm. The signals of DAPI and CLas-Cy3 are the same as described in Figure 2. Data are shown as means ± SEM with at least nine independent biological replications. The significant differences between treatment and controls are indicated by asterisks (Student’s t-test, ***p<0.001).
Figure 6
The juvenile hormone (JH) signaling pathway is regulated by adipokinetic hormone (AKH) signaling pathway and is involved in the increase in fecundity of D. citri induced by Candidatus Liberibacter asiaticus (CLas).
(A) JH titer in the abdomen of CLas + females treated with dsDcAKH for 48 hr. (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 hr. (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 hr. (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. Data are shown as means ± SEM with at least nine independent biological replications.
Figure 7
Mechanisms linking metabolism and reproduction of D. citri induced by Candidatus Liberibacter asiaticus (CLas).
After infection with CLas, the triacylglycerides (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 juvenile hormone (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.
Additional files
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Supplementary file 1
Primer list used for 3’RACE, qRT-PCR, and RNAi analysis.
Related to Method details.
- https://cdn.elifesciences.org/articles/93450/elife-93450-supp1-v1.docx
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Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by ‘Candidatus Liberibacter asiaticus’
eLife 13:RP93450.
https://doi.org/10.7554/eLife.93450.3
