Figure 1.
Starvation decreases mosquito lipid reserves.
The total amount of lipids in the female body significantly increased when mosquitoes were fed 20% sucrose as compared to those fed water alone (0%). Bars represent the means ±SEM of 3 groups of 5 females per treatment. Asterisks denote significant differences between the two treatments for each time point (unpaired t-test, **P<0.01, *P<0.05).
Figure 2.
Starvation decreases JH synthesis and JHAMT transcript levels.
CA-CC complexes were dissected from 4 days old females after 3 days feeding water (0%) or 20% sucrose (20%). A) JH synthesis: groups of 3 glands were incubated for 4 h; JH synthesis was evaluated by HPLC-FD and it is expressed as fmol per CA per hour. Bars represent the means ± S.E.M. of the analysis of 7 independent experiments of 5 groups of 3 glands per treatment. Different letters above the columns indicate significant differences among treatments (unpaired t-test, ***P<0.001). B) JHAMT transcript levels: CA-CC complexes were dissected from 4 days old females fed on water or 20% sucrose. Transcript levels were measured by qPCR and are expressed as copy number of mRNA JHAMT/10,000 copies of rpL32 mRNA. Bars represent the means ± S.E.M. of 10 replicates of groups of 10 CA each (unpaired t-test, *P<0.05).
Figure 3.
Starvation modulates the expression of insulin/TOR pathway genes.
CA-CC complexes were dissected from 4 days old females after 3 days feeding water (0%) or 20% sucrose (20%). Insulin receptor (INSr), forkhead-box 0 (FOXO), 4E-binding protein (4EBP) and target of rapamycin (TOR) transcript levels were measured by qPCR and are expressed as copy number of mRNA gene/10,000 copies of rpL32 mRNA. Bars represent the means ± S.E.M. of 6 replicates of 10 CA each (unpaired t-test, ***P<0.001, **P<0.01, *P<0.05).
Figure 4.
Effects of dsRNA treatment on INSr and FOXO transcript levels.
Newly emerged females were injected with dsYFP, dsINSr or dsFOXO. The effect of the RNAi treatment was evaluated 4 days later. A) Effect of dsINSr treatment. B) Effect of dsFOXO treatment. The expression of INSr and FOXO mRNA in the thorax (without legs and wings) were evaluated by qPCR, and it is expressed as copy number of mRNA INSr or FOXO/10,000 copies of rpL32 mRNA. Bars represent the means ± S.E.M. of 3 independent biological replicates of 3 groups of 3thoraces. Asterisks denote significant difference (unpaired t-test, ***P<0.001,**P<0.01, *P<0.05).
Figure 5.
Effect of RNAi silencing of INSr and FOXO on JH synthesis.
Newly emerged females were injected with dsYFP, dsINSr or dsFOXO. CA-CC complexes were dissected from 4 days old females after 3 days feeding water (0%) or 20% sucrose (20%). Groups of 3 glands were incubated for 4 h and JH synthesis was evaluated by HPLC-FD, and it is expressed as fmol per CA per hour. Bars represent the means ± S.E.M. of the analysis of 6–10 independent experiments of groups of 3 glands per treatment. Different letters above the columns indicate significant differences among treatments (one way ANOVA p<0.05, with Tukey test of multiple comparisons).
Figure 6.
Starvation increases CA insulin sensitivity.
CA-CC complexes were dissected from 4 days old females after 3 days feeding water (0%) or 20% sucrose (20%). Glands were incubated for 4 h in the presence of 17 mM of bovine insulin (+ Ins) or in medium alone. JH synthesis was evaluated by HPLC-FD, and it is expressed as fmol per CA per hour. Bars represent the means ± S.E.M. of the analysis of 3 independent experiments of 3 groups of 3 glands per treatment. Different letters above the columns indicate significant differences among treatments (one way ANOVA p<0.05, with Tukey test of multiple comparisons).
Figure 7.
Starvation effects on insulin signaling components and JH synthesis in the CA of mosquitoes.
This scheme summarizes starvation-related changes of insulin/TOR pathway components and JH synthesis. Molecules in red color are down-regulated (↓), while those in green are upregulated (↑). Previous experiments established the involvement of phosphoinositide 3-kinase (PI3K) and TOR in the transduction of insulin signaling in the CA [13]. Astarvation-dependent decrease of insulin results in an increase of FOXO signaling that promotes activation of transcription of insulin receptor (INSr) and 4E-binding protein (4EBP). Transcripts levels for FOXO increase and mRNAs for JHAMT and TOR decrease. JH synthesis decrease, while increases of 4EBP inhibit translation and increases of INSr enhance insulin sensitivity.