20-Hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Abstract

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again "competent" to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or -24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the -24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the -24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.

Keywords: 20-Hydroxyecdysone; Biosynthesis; Corpora allata; Juvenile hormone; Mosquito.

Fig. 1. Effect of 20E on JH synthesis by Br-CA-CC complexes from “early pupae”

JH biosynthesis by Br-CA-CC complexes incubated for 2 h without (control) and with (+20E) addition of 20E (10⁻⁶ M) was assayed in vitro using the HPLC-FD detection method. Each data point represents the mean ± S.E.M. of 25 independent determinations of groups of 4 Br-CA-CC complexes dissected from −24 h pupae. Asterisks denote significant differences (unpaired t-test; *** P ≤ 0.001).

Fig. 2. Dose-response effect of 20E

The effect on JH synthesis of 20E concentrations ranging from 10⁻⁵ to 10⁻⁷ M were tested on Br-CA-CC complexes dissected from −24 h pupa. Each data point represents the mean ± S.E.M. of at least 25 independent determinations of groups of 4 Br-CA-CC complexes.

Fig. 3. The stimulatory effect of 20E is irreversible

Br-CA-CC were incubated for two consecutive 2 h periods. A) During the first 2 h, Br-CA-CC complexes were incubated either in the presence or absence of 20E (10⁻⁶ M). After 2 h, glands were rinsed in fresh culture medium. B) During the second 2 h period, control and previously treated glands were incubated in fresh culture medium with and without 20E. Each data point represents the mean ± S.E.M. of three independent determinations of three Br-CA-CC complexes. Asterisks denote significant differences (unpaired t-test; *** P ≤ 0.001). Different letters above the columns indicate significant differences among treatments (ANOVA P<0.05, with Tukey’s test for multiple comparison).

Fig. 4. Metabolite pool sizes and enzymatic activities in CA of the early and late pupae

A) Relative levels of endogenous concentrations of FA and MF, and JH biosynthesis rates in CA of early (− 24 h) and late (0 h) pupae. B) Farnesal dehydrogenase (FALDH) and juvenile hormone acid methyl transferase (JHAMT) enzymatic activities in in CA extracts of early (− 24 h) and late (0 h) pupae. Bars represent the means ± SEM of three independent replicates of three groups of 5 CA.

Fig. 5. Effect of stimulation with precursors on JH biosynthesis

The effect of addition of precursors on JH biosynthesis was evaluated on Br-CA-CC complexes incubated in culture medium M-199 alone (Control) or with the addition of 200 μM of FA or MF. A) Br-CA-CC dissected from early pupae (−24 h). B) Br-CA-CC dissected from late pupae (0 h). Each data point represents the means ± S.E.M. of 3 independent biological replicates of three Br-CA-CC complexes. Different letters above the columns indicate significant differences among treatments (ANOVA P<0.05, with Tukey’s test for multiple comparison).

Fig. 6. Effect of 20E on JHAMT enzymatic activity

Br-CA-CC complexes were incubated for 2 h without (Control) and with (+20E) addition of 20E (10⁻⁶ M). Consequently CA-CC extracts were prepared and JHAMT activity was measured. Each data point represents the mean ± S.E.M. of three independent determinations of five CA complexes. Asterisk denotes significant difference (unpaired t-test; *** P ≤ 0.001).

Fig. 7. Effect of denervation on JH synthesis

Br-CA-CC and CA-CC complexes were dissected from early pupae (− 24 h) and incubated in vitro for 2 h. JH biosynthesis was assayed using the HPLC-FD detection method. Each data point represents the mean ± S.E.M. of 3 independent determinations of 4 complexes. Asterisk denotes significant difference (unpaired t-test; *** P ≤ 0.001).