Inhibition of juvenile hormone synthesis in mosquitoes by the methylation inhibitor 3-deazaneplanocin A (DZNep)

Abstract

Juvenile hormone (JH), synthesized by the corpora allata (CA), controls development and reproduction in mosquitoes through its action on thousands of JH-responsive genes. These JH-dependent processes can be studied using tools that increase or decrease JH titers in vitro and in vivo. Juvenile hormone acid methyl transferase (JHAMT) is a critical JH biosynthetic enzyme. JHAMT utilizes the methyl donor S-adenosyl-methionine (SAM) to methylate farnesoic acid (FA) into methyl farnesoate (MF), releasing the product S-adenosyl-L-homocysteine (AdoHcy), which inhibits JHAMT. S-adenosyl-homocysteine hydrolase (SAHH) catalyzes AdoHcy hydrolysis to adenosine and homocysteine, alleviating AdoHcy inhibition of JHAMT. 3-deazaneplanocin A (DZNep), an analog of adenosine, is an inhibitor of SAHH, and an epigenetic drug for cancer therapy. We tested the effect of DZNep on in vitro JH synthesis by CA of mosquitoes. DZNep inhibited JH synthesis in a dose-response fashion. Addition of MF, but not of FA relieved the inhibition, demonstrating a direct effect on JHAMT. In vivo experiments, with addition of DZNep to the sugar ingested by mosquitoes, resulted in a dose-response decrease in JH synthesis and JH hemolymphatic titers, as well as expression of early trypsin, a JH-dependent gene. Our studies suggest that DZNep can be employed to lower JH synthesis and titer in experiments evaluating JH-controlled processes in mosquitoes.

Keywords: Aedes aegypti; Corpora allata; DZNep; Early trypsin; Inhibition; Juvenile hormone synthesis; Methyltransferase; Titer.

Fig 1:. JHAMT and SAHH roles in JH synthesis:

Schema of the last two steps of JH III biosynthesis, with details on the effects of DZNep. JHAMT catalyzes the transfer of a methyl group from AdoMet to FA, to produce MF and AdoHcy. MF is converted to JH III by the action of an epoxidase (EPOX). AdoHcy inhibits JHAMT activity. SAHH is responsible for the reversible hydration of AdoHcy into adenosine (Ado) and homocysteine (Hcy). DZNep inhibits SAHH activity as an adenosine analog. Hcy is methylated to methionine by a methionine synthase (MS). Methionine adenosyltransferase (MAT) creates AdoMet by reacting methionine and ATP.

Fig 2:. DZNep inhibits JH synthesis in vitro in a dose-dependent mode:

A) Addition of DZNep (0.5 µ M) to the incubation media resulted in a significant inhibition of JH synthesis. Bars represent the means ± S.E.M. of 15 independent replicates of incubations of groups of 4 BR-CA-CC complexes. Asterisks denote significant differences (unpaired t-test; *** P ≤ 0.001). B) Six different DZNep concentrations were tested in vitro (0.00005, 0.0005, 0.005, 0.05, 0.25 and 0.5 µM). JH synthesis rates decreased in a dose response manner (r²: 0.9865, P < 0.0001). Points represent the means ± S.E.M. of 4–15 independent replicates of incubations of groups of 4 BR-CA-CC complexes. C) The dynamics of inhibition were studied by comparing JH synthesis in vitro by CA that were incubated for 2h or 4 h in the absence (Co) or presence of DZNep (0.5 µM) (In). Glands incubated with DZNep for 4h still showed increases in JH synthesis when compared to those incubated with DZNep for 2 h. Bars represent the means ± S.E.M. of 8 independent replicates of incubations of groups of 4 BR-CA-CC complexes. Different letters above the bars indicate significant differences among treatments (One way ANOVA, p < 0.05, with Tukey’s test of multiple comparisons).

Fig 3:. Addition of DZNep to the sugar meal resulted in a decrease in JH synthesis and JH hemolymphatic titer.

A) Newly emerged adult female mosquitoes were offered a cotton pad soaked in a 3% sucrose solution in the presence or absence of DZNep (1mM). Three days later, BR-CA-CC complexes were dissected and JH synthesis was evaluated. Bars represent the means ± S.E.M. of 4 independent replicates of incubations of groups of 5 BR-CA-CC complexes. Asterisks denote significant differences (unpaired t-test; ** P ≤ 0.01). B) Hemolymph was collected from the same animals, and JH titer was evaluated. Bars represent the means ± S.E.M. of 4 independent replicates of hemolymph from groups of 5 females. Asterisks denote significant differences (unpaired t-test; * P ≤ 0.05).

Fig 4:. JH inhibition could be reversed by removing DZNep from the diet.

Groups of females were offered for three days a cotton pad soaked in a 3% sucrose solution in the absence (Co 3d) or presence of DZNep (1mM) (In 3d). Some females were offered a sucrose solution for 6 days (Co 6d). To evaluate the effect of removing DZNep from the diet, some females after three days of receiving DZNep, were offered a sucrose solution for another three days (In 3d+3d). Bars represent the means ± S.E.M. of 4 independent replicates of incubations of groups of 4 BR-CA-CC complexes. Different letters above the bars indicate significant differences among treatments (One way ANOVA, p < 0.05, with Tukey’s test of multiple comparisons).

Fig. 5:. DZNep decreases JH synthesis through inhibition of juvenile hormone acid methyl transferase activity:

A) JH synthesis by BR-CA-CC stimulated with MF (200 µM). Co: control, In: with DZNep (5 µM), Co+MF: control + MF, In+MF: DZNep (5 µ M) + MF. B) JH synthesis by BR-CA-CC stimulated with FA (200 µ M). Co: control, In: with DZNep (5 µ M), Co+FA: control + FA, In+FA: DZNep (5 µM) + FA. Bars represent the means ± S.E.M. of 4 independent replicates of incubations of groups of 4 BR-CA-CC complexes. Different letters above the bars indicate significant differences among treatments (One way ANOVA, p < 0.05, with Tukey’s test of multiple comparisons).

Fig 6:. DZNep decreased the expression of the early trypsin gene.

A) Groups of females were offered for three days a cotton pad soaked in a 3% sucrose solution in the absence (Co, black bar) or presence of DZNep (1mM) (In, red bar). Early trypsin mRNA levels in abdomens of treated and untreated females were measured by q-PCR. B) To evaluate the effect of removing DZNep from the diet on ET expression, some females after three days of receiving DZNep, were offered just a sucrose solution for another three days (In 3d+3d), while controls received sucrose for six days (Co 6d). Bars represent the means ± S.E.M. of 8 independent replicates of individual abdomens. Asterisks denote significant differences (unpaired t-test; *** P ≤ 0.001). ns: no significant differences.