Developmental roles of tyrosine metabolism enzymes in the blood-sucking insect Rhodnius prolixus

Abstract

The phenylalanine/tyrosine degradation pathway is frequently described as a catabolic pathway that funnels aromatic amino acids into citric acid cycle intermediates. Previously, we demonstrated that the accumulation of tyrosine generated during the hydrolysis of blood meal proteins in Rhodnius prolixus is potentially toxic, a harmful outcome that is prevented by the action of the first two enzymes in the tyrosine degradation pathway. In this work, we further evaluated the relevance of all other enzymes involved in phenylalanine/tyrosine metabolism in the physiology of this insect. The knockdown of most of these enzymes produced a wide spectrum of distinct phenotypes associated with reproduction, development and nymph survival, demonstrating a highly pleiotropic role of tyrosine metabolism. The phenotypes obtained for two of these enzymes, homogentisate dioxygenase and fumarylacetoacetase, have never before been described in any arthropod. To our knowledge, this report is the first comprehensive gene-silencing analysis of an amino acid metabolism pathway in insects. Amino acid metabolism is exceptionally important in haematophagous arthropods due to their particular feeding behaviour.

Keywords: Chagas disease; Rhodnius prolixus; phenylalanine/tyrosine metabolism; reproduction and development.

Figure 1.

Several distinct phenotypes are produced upon silencing of different tyrosine metabolism enzymes. (a) Tyrosine metabolism pathways. PAH, phenylalanine hydroxylase; TAT, tyrosine aminotransferase; HPPD, 4-hydroxyphenylpyruvate dioxygenase; HgD, homogentisate 1,2-dioxygenase; MAAI, maleylacetoacetate isomerase; FAH, fumarylacetoacetase; TH, tyrosine hydroxylase; DDC, l-DOPA decarboxylase/aromatic l-amino acid decarboxylase; PO, phenoloxidase; DCT, dopachrome tautomerase; DCE, dopachrome conversion enzyme; TβH, tyramine β-hydroxylase. Metabolites are abbreviated as follows: l-DOPA (l-3,4-dihydroxyphenylalanine), DHI (5,6-dihydroxyindole), DHICA (5,6-dihydroxyindole-2-carboxylic acid). The image was modified from Sterkel et al. [3]. (b) The number of eggs laid by R. prolixus females in which enzymes of the Phe/Tyr degradation pathway and TH were silenced by dsRNA injection. (c) Hatching rate. (d) Survival of first-stage nymphs that hatched from eggs laid by females injected with dsRNA. (e) Fourth-stage nymph survival after injection with dsRNA. Cyan dots represent insects that died as fifth-stage nymphs, red dots represent insects that died as fourth-stage nymphs and blue dots represent insects that died during the ecdysis process. Dotted horizontal lines show the ecdysis period. At least two independent experiments were performed for each target gene, each with n = 8–12 insects per experimental group. The data from multiple experiments were combined into a single graph. The data were plotted as the mean ± s.e.m. Two-way ANOVA was performed to evaluate differences between the experimental and control groups in (a,b). The log-rank (Kaplan–Meier) test was used to evaluate significant differences in survival between the experimental and control groups in (d,e) (****p < 0.0001).

Figure 2.

Silencing of PAH, FAH and TH interferes with embryo development and hatching. (a) Control egg (dsMAL). (b) Egg from female injected with dsPAH. (c) Egg from female injected with dsFAH. (d) Egg from female injected with dsTH. Images were taken 20–21 days after the eggs were laid.

Figure 3.

Supplementation with exogenous tyrosine to R. prolixus females does not rescue or alleviate the PAH silencing phenotype. Control (MAL) and PAH-silenced R. prolixus females received two injections of 50 nmol of tyrosine at day 1 before blood meal and at day 2 PBM. At least two independent experiments were performed for each target gene. The data from multiple experiments were combined into a single graph. The data were plotted as the mean ± s.e.m. Two-way ANOVA was performed to evaluate differences between the experimental and control groups in (a,b). The log-rank (Kaplan–Meier) test was used to evaluate significant differences between the experimental and control groups in panel (c) (****p < 0.0001).

Figure 4.

Administration of l-DOPA decarboxylase/aromatic l-amino acid decarboxylase (DDC) inhibitor carbidopa to R. prolixus females delays oviposition and decreases the hatching of embryos. (a) Oviposition. (b) Egg hatching. (c) First-stage nymph survival. Five microlitres of carbidopa 2.5 mM dissolved in H₂O were injected into the thorax on day 1 BBM and day 2 PBM, or on day 2 PBM and day 5 PBM. Two independent experiments were performed for each carbidopa treatment, each with n = 8–12 insects per experimental group. The data from both experiments were combined into a single graph. The data were plotted as the mean ± s.e.m. Two-way ANOVA was performed to evaluate differences between the experimental and control groups in (a,b). The log-rank (Kaplan–Meier) test was used to evaluate significant differences between the experimental and control groups in (c) (**p < 0.01).

Figure 5.

Silencing of FAH affects embryo development. Shown are phenotypes observed in FAH-silenced first-stage nymphs (N1) that hatched from eggs laid by females injected with dsFAH, revealing an important role of this enzyme in embryo morphogenetic events. (a) Dorsal view of control N1. (b) Ventral view of control N1. (c) Dorsal view of FAH-silenced N1. (d) Ventral view of FAH-silenced N1.