The tryptophan oxidation pathway in mosquitoes with emphasis on xanthurenic acid biosynthesis

Oxidation of tryptophan to kynurenine and 3-hydroxykynurenine (3-HK) is the major catabolic pathway in mosquitoes. However, 3-HK is oxidized easily under physiological conditions, resulting in the production of reactive radical species. To overcome this problem, mosquitoes have developed an efficient mechanism to prevent 3-HK from accumulating by converting this chemically reactive compound to the chemically stable xanthurenic acid. Interestingly, 3-HK is a precursor for the production of compound eye pigments during the pupal and early adult stages; consequently, mosquitoes need to preserve and transport 3-HK for compound eye pigmentation in pupae and adults. This review summarizes the tryptophan oxidation pathway, compares and contrasts the mosquito tryptophan oxidation pathway with other model species, and discusses possible driving forces leading to the functional adaptation and evolution of enzymes involved in the mosquito tryptophan oxidation pathway.     

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

Precursors of pattern specific ommatin in red wing scales of the polyphenic butterfly Araschnia levana L.: Haemolymph tryptophan and 3-hydroxykynurenine

In the seasonally diphenic butterfly Araschnia levana¹⁴C-labelled tryptophan and 3-hydroxykynurenine, the principal precursors of ommochromes, injected into young pupae caused a pattern specific radiolabel of mature red scales. [¹⁴C]glucose and [³⁵S]methionine also labelled red scales but only when injected shortly before or during the time of pigment synthesis in the wing. In developing non-diapause pupae contents of 3-hydroxykynurenine increased until an abrupt decrease when pigments appeared in the wings. In diapausing pupae 3-hydroxykynurenine remained low but increased after injection of 20-hydroxyecdysone which induced pupal-adult development. Supply of wing scale cells with ommochrome precursors via the haemolymph was analysed after injection of [³H]tryptophan. In developing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine increased at the time of wing pigment formation and decreased shortly before adult emergence. In diapausing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine were low compared to non-diapause pupae but increased at the time of wing pigment formation after injection of 20-hydroxyecdysone. Isolated wings incubated in Grace's medium containing [¹⁴C]tryptophan and [¹⁴C]3-hydroxykynurenine incorporated radiolabel specifically into red portions of the wing colour pattern due to labelling of ommatin. Incorporation into red wing areas occurred specifically depending on different colour patterns of the spring- and the summer-morph.The results demonstrate that both tryptophan as well as 3-hydroxykynurenine are delivered via the haemolymph, and both can serve as precursors of ommatin formation in the scale cells. Therefore, the complete set of enzymes for the tryptophan-ommatin pathway is present in scale-forming cells.     

Tryptophan metabolism during development of the stick insect,Carausius morosus Br. tissue distribution and interrelationships of metabolites of the kynurenine pathway

Summary During larval development ofCarausius morosus kynurenic acid is the major end product of tryptophan metabolism. Tryptophan and kynurenic acid have been found in the fat body, haemolymph and gut contents but only traces of kynurenine have been detected. The ommochromes ommin and xanthommatin are formed in relatively small amounts in the epidermis during larval development. 3-hydroxykynurenine was found only in the epidermis, the site of ommochrome deposition.During larval development, the amount of free tryptophan increases with body dry weight. The amount of kynurenic acid excreted also corresponds to the increase of body weight but is significantly reduced in the faeces of adults. This is related to a high tryptophan content of yolk proteins. The concentration of tryptophan in the haemolymph decreases immediately before ecdysis, whereas that in the gut increases during this time and falls sharply at the start of ecdysis.     

Interference of some tryptophan metabolites in the formation of melanin in vitro

Melanin (eumelanin) is commonly produced in mammals starting from tyrosine and/or 3,4-dioxyphenylalanine (DOPA) under the action of tyrosinase. 3-Hydroxyanthranilic acid and 3-hydroxykynurenine are intermediates occurring in the kynurenine pathway of tryptophan catabolism. In this paper, we show that these substances can interfere in melanin formation in vitro when tyrosine or DOPA is oxidized by molecular oxygen under catalysis by tyrosinase. In particular, when 3-hydroxyanthranilic acid is present, a brown and apparently water-soluble pigment is formed, whereas the typical eumelanin granules seem to become more and more rare as the concentration of 3-hydroxyanthranilic acid increases. Also in the presence of the latter, the rate of tyrosine and/or DOPA consumption decreases. A very complicated (13)C-NMR spectrum indicates the high complexity of the reaction. This involves both the true melanin precursor(s) and the tryptophan metabolite, even if with peculiar mechanism and kinetics. When 3-hydroxykynurenine is substituted for 3-hydroxyanthranilic acid the reaction leads to reddish pigments whereas xanthommatins (the typical oxidation products of 3-hydroxykynurenine) are absent. A possible relationship between some dischromic pathologies and tryptophan metabolic disorders is suggested.     

Discovery of 1,2,4-oxadiazole derivatives as a novel class of noncompetitive inhibitors of 3-hydroxykynurenine transaminase (HKT) from Aedes aegypti

The mosquito Aedes aegypti is the vector of arboviruses such as Zika, Chikungunya, dengue and yellow fever. These infectious diseases have a major impact on public health. The unavailability of effective vaccines or drugs to prevent or treat most of these diseases makes vector control the main form of prevention. One strategy to promote mosquito population control is the use of synthetic insecticides to inhibit key enzymes in the metabolic pathway of these insects, particularly during larval stages. One of the main targets of the kynurenine detoxification pathway in mosquitoes is the enzyme 3-hydroxykynurenine transaminase (HKT), which catalyzes the conversion of 3-hydroxykynurenine (3-HK) into xanthurenic acid (XA). In this work, we report eleven newly synthesized oxadiazole derivatives and demonstrate that these compounds are potent noncompetitive inhibitors of HKT from Ae. aegypti. The present data provide direct evidence that HKT can be explored as a molecular target for the discovery of novel larvicides against Ae. aegypti. More importantly, it ensures that structural information derived from the HKT 3D-structure can be used to guide the development of more potent inhibitors.     

Crystal structures of Aedes aegypti alanine glyoxylate aminotransferase

Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75A high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1A resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.     

Ommochromes in invertebrates: biochemistry and cell biology

Ommochromes are widely occurring coloured molecules of invertebrates, arising from tryptophan catabolism through the so‐called Tryptophan → Ommochrome pathway. They are mainly known to mediate compound eye vision, as well as reversible and irreversible colour patterning. Ommochromes might also be involved in cell homeostasis by detoxifying free tryptophan and buffering oxidative stress. These biological functions are directly linked to their unique chromophore, the phenoxazine/phenothiazine system. The most recent reviews on ommochrome biochemistry were published more than 30 years ago, since when new results on the enzymes of the ommochrome pathway, on ommochrome photochemistry as well as on their antiradical capacities have been obtained. Ommochromasomes are the organelles where ommochromes are synthesised and stored. Hence, they play an important role in mediating ommochrome functions. Ommochromasomes are part of the lysosome‐related organelles (LROs) family, which includes other pigmented organelles such as vertebrate melanosomes. Ommochromasomes are unique because they are the only LRO for which a recycling process during reversible colour change has been described. Herein, we provide an update on ommochrome biochemistry, photoreactivity and antiradical capacities to explain their diversity and behaviour both in vivo and in vitro. We also highlight new biochemical techniques, such as quantum chemistry, metabolomics and crystallography, which could lead to major advances in their chemical and functional characterisation. We then focus on ommochromasome structure and formation by drawing parallels with the well‐characterised melanosomes of vertebrates. The biochemical, genetic, cellular and microscopic tools that have been applied to melanosomes should provide important information on the ommochromasome life cycle. We propose LRO‐based models for ommochromasome biogenesis and recycling that could be tested in the future. Using the context of insect compound eyes, we finally emphasise the importance of an integrated approach in understanding the biological functions of ommochromes.     

Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine.

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxykynurenine, and hydroxyl radicals, formed via H(2)O(2), may also be implicated [Okuda, S., Nishiyama, N., Saito, H. , and Katsuki, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydroxykynurenine, also results in the formation of highly reactive quinonimines. Thus, one possible consequence of 3-hydroxykynurenine oxidation may be covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter. In this work, we demonstrate that 3-hydroxykynurenine can bind to protein amino groups and, further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colored and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of this lens protein in the presence of 3-hydroxykynurenine. Similar posttranslational modifications of lens proteins accompany cataract formation, and knowledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerative conditions in which oxidation of such aminophenols is implicated.     

Eye pigments of the blood-sucking insect, Triatoma infestans Klug (Hemiptera, Reduviidae).

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectrophotometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, melanins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black- and red-eyed specimens of T. infestans for other characteristics.     

Brain development in the yellow fever mosquito Aedes aegypti: a comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster

Considerable effort has been directed towards understanding the organization and function of peripheral and central nervous system of disease vector mosquitoes such as Aedes aegypti. To date, all of these investigations have been carried out on adults but none of the studies addressed the development of the nervous system during the larval and pupal stages in mosquitoes. Here, we first screen a set of 30 antibodies, which have been used to study brain development in Drosophila, and identify 13 of them cross-reacting and labeling epitopes in the developing brain of Aedes. We then use the identified antibodies in immunolabeling studies to characterize general neuroanatomical features of the developing brain and compare them with the well-studied model system, Drosophila melanogaster, in larval, pupal, and adult stages. Furthermore, we use immunolabeling to document the development of specific components of the Aedes brain, namely the optic lobes, the subesophageal neuropil, and serotonergic system of the subesophageal neuropil in more detail. Our study reveals prominent differences in the developing brain in the larval stage as compared to the pupal (and adult) stage of Aedes. The results also uncover interesting similarities and marked differences in brain development of Aedes as compared to Drosophila. Taken together, this investigation forms the basis for future cellular and molecular investigations of brain development in this important disease vector.     

Effects of variations in a formulated protein meal on the fecundity and fertility of female mosquitoes

Abstract. A formulated protein meal developed by Kogan (1990) for adult female Aedes aegypti mosquitoes was evaluated and modified to increase egg and pupal yield. A vigorous laboratory colony was maintained with the females fed exclusively on this dietary formula for about twenty-five generations over more than 2 years. Extra modifications were made to produce a diet suitable for Anopheles arabiensis and Anopheles stephensi females to produce eggs. Both formulations contain bovine albumin, haemoglobin and globulin in a ringer based solution, plus ATP as a phagostimulant for Ae.aegypti. Compared to Kogan's original, our Aedes formula doubled the production of pupae per female after a single meal, although the yield was still significantly lower than from mosquitoes fed on animal hosts or defibrinated pig blood. In varying the proportions of different constituents during attempts to optimize the formula, no relationship was found between total protein content (within the range 80–220 mg/ml) and fecundity, percentage hatch or pupal yield of Ae.aegypti. Equivocal results were found when an isoleucine supplement was added to the formula.     

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase

Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic intermediate of the tryptophan degradation pathway, elicits a remarkable oxidative stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more stable compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gametocyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR-based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expression analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purification of a fully active recombinant version of the protein has been developed. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5'-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission-blocking drugs or effective insecticides.     

A general mechanism of polypeptide cross-linking by 3-hydroxykynurenine

Abstract

The human lens contains a group of fluorescent compounds, derived from tryptophan, which act to absorb UV light in the 300–400 nm region of the spectrum.¹ The major component is the glucoside of 3-hydroxykynurenine (3HK), 3-hydroxykynurenine glucoside (3HKG).²In the lens, 3HKG represents a unique pathway of tryptophan metabolism. Smaller amounts of kynurenine and 3HK have been detected in human lens extracts.^3,4 . More recently, a new UV-filter compound derived from tryptophan, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (AHBG), was identified, and constitutes the second most abundant UV-filter in the human lens.⁵     

Water Level Flux in Household Containers in Vietnam - A Key Determinant of Aedes aegypti Population Dynamics

We examined changes in the abundance of immature Aedes aegypti at the household and water storage container level during the dry-season (June-July, 2008) in Tri Nguyen village, central Vietnam. We conducted quantitative immature mosquito surveys of 171 containers in the same 41 households, with replacement of samples, every two days during a 29-day period. We developed multi-level mixed effects regression models to investigate container and household variability in pupal abundance. The percentage of houses that were positive for I/II instars, III/IV instars and pupae during any one survey ranged from 19.5-43.9%, 48.8-75.6% and 17.1-53.7%, respectively. The mean numbers of Ae. aegypti pupae per house ranged between 1.9-12.6 over the study period. Estimates of absolute pupal abundance were highly variable over the 29-day period despite relatively stable weather conditions. Most variability in pupal abundance occurred at the container rather than the household level. A key determinant of Ae. aegypti production was the frequent filling of the containers with water, which caused asynchronous hatching of Ae. aegypti eggs and development of cohorts of immatures. We calculated the probability of the water volume of a large container (>500L) increasing or decreasing by ≥20% to be 0.05 and 0.07 per day, respectively, and for small containers (<500L) to be 0.11 and 0.13 per day, respectively. These human water-management behaviors are important determinants of Ae. aegypti production during the dry season. This has implications for choosing a suitable Wolbachia strain for release as it appears that prolonged egg desiccation does not occur in this village.     

Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

A biochemical study of the scarlet eye-color mutant of Drosophila melanogaster

3-Hydroxykynurenine is virtually absent from st larvae but accumulates during adult development in the puparium. Over the period of adult emergence, the accumulated 3-hydroxykynurenine is excreted so that st adults contain none. Larvae of st fed on tryptophan-C ¹⁴ medium produce labeled 3-hydroxykynurenine, at a reduced rate, perhaps, compared to wild type. Xanthurenic acid levels in st pupae are similar to those in wild type. Thus the failure of st larvae to accumulate 3-hydroxykynurenine does not seem to be due either to an inability to synthesize this compound or to an excessive rate of its conversion to xanthurenic acid. Rather, it appears that the mechanism of 3-hydroxykynurenine storage during larval life is defective, so that this compound is excreted at an abnormally high rate. The inability of the pigment cells of the eyes of st to synthesize xanthommatin may result from a similar defect in their ability to take up or store 3-hydroxykynurenine.