Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes

Abstract

We demonstrate the presence of an alternate metabolic pathway for urea synthesis in Aedes aegypti mosquitoes that converts uric acid to urea via an amphibian-like uricolytic pathway. For these studies, female mosquitoes were fed a sucrose solution containing (15)NH4Cl, [5-(15)N]-glutamine, [(15)N]-proline, allantoin, or allantoic acid. At 24 h after feeding, the feces were collected and analyzed in a mass spectrometer. Specific enzyme inhibitors confirmed that mosquitoes incorporate (15)N from (15)NH4Cl into [5-(15)N]-glutamine and use the (15)N of the amide group of glutamine to produce labeled uric acid. More importantly, we found that [(15)N2]-uric acid can be metabolized to [(15)N]-urea and be excreted as nitrogenous waste through an uricolytic pathway. Ae. aegypti express all three genes in this pathway, namely, urate oxidase, allantoinase, and allantoicase. The functional relevance of these genes in mosquitoes was shown by feeding allantoin or allantoic acid, which significantly increased unlabeled urea levels in the feces. Moreover, knockdown of urate oxidase expression by RNA interference demonstrated that this pathway is active in females fed blood or (15)NH4Cl based on a significant increase in uric acid levels in whole-body extracts and a reduction in [(15)N]-urea excretion, respectively. These unexpected findings could lead to the development of metabolism-based strategies for mosquito control.

Fig. 1.

Effect of 80 mM ¹⁵NH₄Cl, [5-¹⁵N]-Gln, or [¹⁵N]-Pro on urea synthesis in Ae. aegypti mosquitoes. [¹⁵N]-urea concentrations were measured in the mosquito feces 24 h after feeding with 80 mM¹⁵NH₄Cl, [5-¹⁵N]-Gln, or [¹⁵N]-Pro. Data are presented as mean ± SE of three independent samples. *, P < 0.05 when compared with ¹⁵NH₄Cl by ANOVA.

Fig. 2.

Time course of whole-body labeled amino acids from Ae. aegypti after feeding with 30 or 80 mM [¹⁵N]-Pro. (A) [¹⁵N]-Pro. (B) [5-¹⁵N]-Gln. Data are presented as mean ± SE of three independent samples. *, P < 0.05; **, P < 0.01 [when compared with 0 (immediately after feeding) by ANOVA].

Fig. 3.

Effect of specific inhibitors on [¹⁵N]-urea synthesis. [¹⁵N]-urea concentrations were measured in the mosquito feces 24 h after feeding with 80 mM ¹⁵NH₄Cl and 20 mM methionine sulfoximine (A) or 80 mM [5-¹⁵N]-Gln and 1 mM allopurinol (B). Control mosquitoes (0 mM) were fed in the absence of inhibitor. Data are presented as mean ± SE of three independent samples. *, P < 0.05 (when compared with 0 mM by Student's t test).

Fig. 4.

Effect of allantoin and allantoic acid on urea synthesis. Urea concentrations were measured in the mosquito feces at 24 h after feeding. (A) After feeding with 0, 10, and 30 mM allantoin. (B) After feeding with 0, 10, and 30 mM allantoic acid. Control mosquitoes (0 mM) were fed only on 3% sucrose. Data are presented as mean ± SE of three independent samples. *, P < 0.05; **, P < 0.01 (when compared with 0 mM by ANOVA).

Fig. 5.

The patterns of UO, ALN, and ALLC gene expression in Ae. aegypti females (AaUO, AaALN, and AaALLC). Shown is the relative abundance of mRNA in FB and MT tissues of mosquitoes after feeding with a blood meal (A–F) or after feeding with 80 mM ¹⁵NH₄Cl (G and H). The mRNA levels were normalized according to the mRNA level of the S7 ribosomal protein. Sucrose-fed females (SF) were fed only on 3% sucrose. Data are presented as mean ± SE of three or five independent samples. *, P < 0.05; **, P < 0.01 (when compared with SF by ANOVA).

Fig. 6.

Concentration of urea, allantoin, and allantoic acid in Ae. aegypti feces. Females were fed on blood meal, and the feces were analyzed at 24, 48, and 72 h after feeding. Data are presented as mean ± SE of five independent samples. *, P < 0.05 (when compared with 24 h by ANOVA).

Fig. 7.

Effect of UO knockdown on gene expression. Ae. aegypti mosquitoes were injected with dsRNAi-Fluc or dsRNAi-UO and then fed with a blood meal or 80 mM ¹⁵NH₄Cl. (A) Relative abundance of UO mRNA in tissues of injected mosquitoes at 24 h after feeding with a blood meal. The mRNA levels were normalized according to mRNA level of the S7 ribosomal protein. (B) Total uric acid concentration in whole-body mosquito at 24 and 48 h after feeding with a blood meal. (C) Total uric acid concentration in whole-body mosquito at 1 and 24 h after feeding with ¹⁵NH₄Cl. (D) [¹⁵N]-urea concentrations in the mosquito feces at 24 h after feeding with ¹⁵NH₄Cl. Data are presented as mean ± SE of three independent samples. *, P < 0.05; **, P < 0.01 (when compared with dsRNAi-Fluc by Student's t test).

Fig. 8.

Proposed metabolic pathway for uric acid degradation and urea synthesis in Ae. aegypti mosquitoes. The labeled nitrogen atoms are shown in red.