RESUMO
BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.
Assuntos
Aedes , Mosquitos Vetores , Humanos , Animais , Genótipo , Mosquitos Vetores/genética , Heterozigoto , Aedes/genéticaRESUMO
We previously demonstrated that Aedes aegypti pyruvate kinase (AaPK) plays a key role in the regulation of both carbon and nitrogen metabolism in mosquitoes. To further elucidate whether AaPK can be post-translationally regulated by Ae. aegypti sirtuin 2 (AaSirt2), an NAD+-dependent deacetylase that catalyzes the removal of acetyl groups from acetylated lysine residues, we conducted a series of analysis in non-starved and starved female mosquitoes. Transcriptional and protein profiles of AaSirt2, analyzed by qPCR and western blots, indicated that the AaSirt2 is differentially modulated in response to sugar or blood feeding in mosquito tissues dissected at different times during the first gonotrophic cycle. We also found that AaSirt2 is localized in both cytosolic and mitochondrial cellular compartments of fat body and thorax. Multiple lysine-acetylated proteins were detected by western blotting in both cellular compartments. Furthermore, western blotting of immunoprecipitated proteins provided evidence that AaPK is lysine-acetylated and bound with AaSirt2 in the cytosolic fractions of fat body and thorax from non-starved and starved females. In correlation with these results, we also discovered that RNAi-mediated knockdown of AaSirt2 in the fat body of starved females significantly decreased AaPK protein abundance. Notably, survivorship of AaSirt2-deficient females maintained under four different nutritional regimens was not significantly affected. Taken together, our data reveal that AaPK is post-translationally regulated by AaSirt2.
Assuntos
Aedes , Feminino , Animais , Aedes/metabolismo , Piruvato Quinase/metabolismo , Sirtuína 2/metabolismo , Lisina/metabolismo , Interferência de RNARESUMO
Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, the rate-limiting first step for de novo polyamine biosynthesis. Previously, we reported that genetic knockdown of xanthine dehydrogenase 1 (XDH1)-a gene encoding the enzyme involved in the last two steps of uric acid synthesis-causes an increase in ODC transcript levels in fat body of blood-fed Aedes aegypti mosquitoes, suggesting a crosstalk at molecular level between XDH1 and ODC during nitrogen disposal. To further investigate the role of ODC in nitrogen metabolism, we conducted several biochemical and genetic analyses in sugar- and blood-fed A. aegypti females. Distinct ODC gene and protein expression patterns were observed in mosquito tissues dissected during the first gonotrophic cycle. Both pharmacological and RNA interference-mediated knockdown of ODC negatively impacted mosquito survival, disrupted nitrogen waste disposal, delayed oviposition onset, and decreased fecundity in vitellogenic blood-fed females. A lag in the expression of two major digestive serine proteases, a reduction of blood meal digestion in the midgut, and a decrease in vitellogenin yolk protein uptake in ovarian follicles were observed by western blots in ODC-deficient females. Moreover, genetic silencing of ODC showed a broad transcriptional modulation of genes encoding proteins involved in multiple metabolic pathways in mosquito fat body, midgut, and Malpighian tubules prior to and after blood feeding. All together, these data demonstrate that ODC plays an essential role in mosquito metabolism, and that ODC crosstalks with multiple genes and proteins to prevent deadly nitrogen perturbations in A. aegypti females.
Assuntos
Aedes , Animais , Feminino , Nitrogênio/metabolismo , Ornitina , Ornitina Descarboxilase/genética , OviposiçãoRESUMO
Nearly half a million people die annually due to mosquito-borne diseases. Despite aggressive mosquito population-control efforts, current strategies are limited in their ability to control these vectors. A better understanding of mosquito metabolism through modern approaches can contribute to the discovery of novel metabolic targets and/or regulators and lead to the development of better mosquito-control strategies. Currently, cutting-edge technologies such as gas or liquid chromatography-mass spectrometry-based metabolomics are considered 'mature technologies' in many life-science disciplines but are still an emerging area of research in medical entomology. This review primarily discusses recent developments and progress in the application of mass spectrometry-based metabolomics to answer multiple biological questions related to mosquito metabolism.
Assuntos
Culicidae/metabolismo , Espectrometria de Massas , Metabolômica , Animais , Culicidae/genética , Metaboloma/fisiologia , Metabolômica/instrumentação , Metabolômica/tendênciasRESUMO
A recent in vitro characterization of a recombinant pyruvate kinase (PK) from Aedes aegypti mosquitoes demonstrated that the enzyme is uniquely regulated by multiple allosteric effectors. Here, we further explored PK gene and protein expression, and enzymatic activity in key metabolic tissues of mosquitoes maintained under different nutritional conditions. We also studied the metabolic effects of PK depletion using several techniques including RNA interference and mass spectrometry-based stable-isotope tracing. Transcriptional analysis showed a dynamic post-feeding PK mRNA expression pattern within and across mosquito tissues, whereas corresponding protein levels remained stable throughout the time course analyzed. Nevertheless, PK activity significantly differed in the fat body of sucrose-, blood-fed, and starved mosquitoes. Genetic silencing of PK did not alter survival in blood-fed females maintained on sucrose. However, an enhanced survivorship was observed in PK-deficient females maintained under different nutritional regimens. Our results indicate that mosquitoes overcame PK deficiency by up-regulating the expression of genes encoding NADP-malic enzyme-1, phosphoenolpyruvate carboxykinase-1, phosphoglycerate dehydrogenase and glutamate dehydrogenase, and by decreasing glucose oxidation and metabolic pathways associated with ammonia detoxification. Taken together, our data demonstrate that PK confers to A. aegypti a metabolic plasticity to tightly regulate both carbon and nitrogen metabolism.
Assuntos
Aedes/genética , Isótopos de Carbono/análise , Expressão Gênica , Proteínas de Insetos/genética , Piruvato Quinase/genética , Aedes/enzimologia , Aedes/metabolismo , Animais , Proteínas de Insetos/deficiência , Proteínas de Insetos/metabolismo , Espectrometria de Massas , Piruvato Quinase/deficiência , Piruvato Quinase/metabolismo , Interferência de RNARESUMO
Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of â¼57â¯kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.
Assuntos
Aedes/enzimologia , Frutosedifosfatos/química , Glucose-6-Fosfato/química , Proteínas de Insetos/química , Piruvato Quinase/química , Aedes/genética , Regulação Alostérica/fisiologia , Processamento Alternativo/fisiologia , Animais , Feminino , Frutosedifosfatos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfato/metabolismo , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Cinética , Domínios Proteicos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In Aedes aegypti females, the ammonia released during blood meal digestion is partially metabolized to facilitate the disposal of excess nitrogen. In this study, we used low- and high-resolution liquid chromatography-mass spectrometry (LC/MS) techniques to investigate the role of glucose during ammonia detoxification. Mosquitoes were fed a blood meal supplemented with [1,2-13C2]glucose, and downstream metabolites were measured for 24 h. Quantification of [13C] amino acids in the entire mosquito body was conducted without sample derivatization using selected reaction monitoring of mass transitions that are indicative of the structural position of [13C] atom incorporation. Identification of unlabeled and [13C] isotopologs of 43 compounds, including amino acids, amino acid derivatives, and organic acids, was performed by high-resolution LC/MS techniques. Blood-fed mosquitoes synthesized [13C] metabolites in mainly 2 carbon positions from [1,2-13C2]glucose. [13C2]Ala and [13C2]Pro were the most abundant and rapidly labeled amino acids synthesized. Additional [13C] amino acids, [13C] amino acid derivatives, and [13C] organic acids in 1 or 2 carbon positions were also identified. Two kinetic routes were proposed based on the incorporation of a [13C] atom at position 1 in specific amino acids. Our findings provide evidence that glucose is used for ammonia detoxification and [13C] uric acid synthesis through multiple metabolic pathways, uncovering a metabolic link at the carbon atomic level in ammonia metabolism of A. aegypti-Horvath, T. D., Dagan, S., Lorenzi, P. L., Hawke, D. H., Scaraffia, P. Y. Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes.
Assuntos
Aedes/metabolismo , Amônia/metabolismo , Carbono/metabolismo , Aminoácidos/metabolismo , Animais , Isótopos de Carbono/metabolismo , Cromatografia Líquida , Feminino , Glucose/metabolismo , Isótopos , Espectrometria de Massas , Redes e Vias Metabólicas , Metabolômica , Modelos Biológicos , Nitrogênio/metabolismoRESUMO
Aedesaegypti has 2 genes encoding xanthine dehydrogenase (XDH). We analyzed XDH1 and XDH2 gene expression by real-time quantitative PCR in tissues from sugar- and blood-fed females. Differential XDH1 and XDH2 gene expression was observed in tissues dissected throughout a time course. We next exposed females to blood meals supplemented with allopurinol, a well-characterized XDH inhibitor. We also tested the effects of injecting double-stranded RNA (dsRNA) against XDH1, XDH2, or both. Disruption of XDH by allopurinol or XDH1 by RNA interference significantly affected mosquito survival, causing a disruption in blood digestion, excretion, oviposition, and reproduction. XDH1-deficient mosquitoes showed a persistence of serine proteases in the midgut at 48 h after blood feeding and a reduction in the uptake of vitellogenin by the ovaries. Surprisingly, analysis of the fat body from dsRNA-XDH1-injected mosquitoes fell into 2 groups: one group was characterized by a reduction of the XDH1 transcript, whereas the other group was characterized by an up-regulation of several transcripts, including XDH1, glutamine synthetase, alanine aminotransferase, catalase, superoxide dismutase, ornithine decarboxylase, glutamate receptor, and ammonia transporter. Our data demonstrate that XDH1 plays an essential role and that XDH1 has the potential to be used as a metabolic target for Ae.aegypti vector control.-Isoe, J., Petchampai, N., Isoe, Y. E., Co, K., Mazzalupo, S., Scaraffia, P. Y. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.
Assuntos
Aedes/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Inativação Gênica , Xantina Desidrogenase/metabolismo , Aedes/genética , Alopurinol/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Controle de Mosquitos , Nitrogênio/metabolismo , Oviposição/efeitos dos fármacos , Óvulo , Sacarose , Xantina Desidrogenase/classificação , Xantina Desidrogenase/genéticaRESUMO
To better understand the mechanisms responsible for the success of female mosquitoes in their disposal of excess nitrogen, we investigated the role of alanine aminotransferase (ALAT) in blood-fed Aedes aegypti. Transcript and protein levels from the 2 ALAT genes were analyzed in sucrose- and blood-fed A. aegypti tissues. ALAT1 and ALAT2 exhibit distinct expression patterns in tissues during the first gonotrophic cycle. Injection of female mosquitoes with either double-stranded RNA (dsRNA)-ALAT1 or dsRNA ALAT2 significantly decreased mRNA and protein levels of ALAT1 or ALAT2 in fat body, thorax, and Malpighian tubules compared with dsRNA firefly luciferase-injected control mosquitoes. The silencing of either A. aegypti ALAT1 or ALAT2 caused unexpected phenotypes such as a delay in blood digestion, a massive accumulation of uric acid in the midgut posterior region, and a significant decrease of nitrogen waste excretion during the first 48 h after blood feeding. Concurrently, the expression of genes encoding xanthine dehydrogenase and ammonia transporter (Rhesus 50 glycoprotein) were significantly increased in tissues of both ALAT1- and ALAT2-deficient females. Moreover, perturbation of ALAT1 and ALAT2 in the female mosquitoes delayed oviposition and reduced egg production. These novel findings underscore the efficient mechanisms that blood-fed mosquitoes use to avoid ammonia toxicity and free radical damage.-Mazzalupo, S., Isoe, J., Belloni, V., Scaraffia, P. Y. Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase.
Assuntos
Aedes/enzimologia , Alanina Transaminase/metabolismo , Corpo Adiposo/metabolismo , Nitrogênio/metabolismo , Aedes/genética , Animais , Digestão/fisiologia , Feminino , RNA de Cadeia Dupla/metabolismoRESUMO
Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A. stephensi and Ae. aegypti Akt in the fat body of transgenic mosquitoes led to activation of the downstream signaling molecules forkhead box O (FOXO) and p70 S6 kinase in a tissue and blood meal-specific manner. In both species, increased Akt signaling in the fat body after blood feeding significantly increased adult survivorship relative to nontransgenic sibling controls. In A. stephensi, survivorship was increased by 15% to 45%, while in Ae. aegypti, it increased 14% to 47%. Transgenic mosquitoes fed only sugar, and thus not expressing active Akt, had no significant difference in survivorship relative to nontransgenic siblings. Expression of active Akt also increased expression of fat body vitellogenin, but the number of viable eggs did not differ significantly between transgenic and nontransgenic controls. This work demonstrates a novel mechanism of enhanced survivorship through increased Akt signaling in the fat bodies of multiple mosquito genera and provides new tools to unlock the molecular underpinnings of aging in eukaryotic organisms.
Assuntos
Aedes/metabolismo , Anopheles/metabolismo , Corpo Adiposo/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Aedes/genética , Aedes/crescimento & desenvolvimento , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Animais Geneticamente Modificados , Anopheles/genética , Anopheles/crescimento & desenvolvimento , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Insetos/genética , Longevidade/genética , Longevidade/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Reprodução/genética , Reprodução/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Especificidade da Espécie , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMO
BACKGROUND: It was previously demonstrated that alanine aminotransferase (ALAT, EC 2.6.1.2) participates in maintaining the alanine-proline cycle between flight muscles and fat body during Aedes aegypti flight. ALAT is also actively involved in the metabolism of ammonia in A. aegypti. Here, we investigated the survival and behavioral costs of ALAT inhibition in A. aegypti females to better understand the role of ALAT in blood-fed mosquitoes. METHODS: We analyzed how A. aegypti female mosquitoes respond to blood meals supplemented with 0, 2.5, 5 and 10 mM L-cycloserine, a well-known inhibitor of ALAT in animals. Mosquitoes were also exposed to blood meals supplemented with L-cycloserine and different concentrations of glucose (0, 10 and 100 mM). Additionally, the effects of ALAT inhibitor and glucose in mosquitoes starved for 24 or 48 h were investigated. Survival and behavioral phenotypes were analyzed during a time course (1, 2, 4, 6, 12, 24, 48 and 72 h after feeding). RESULTS: L-cycloserine at 10 mM resulted in high mortality relative to control, with an acute effect during the first 6 h after treatment. A significant decrease in the number of active mosquitoes coinciding with an increase in futile wing fanning during the first 24 h was observed at all inhibitor concentrations. A high occurrence of knockdown phenotype was also recorded at this time for both 5 and 10 mM L-cycloserine. The supplementation of glucose in the blood meal amplified the effects of the ALAT inhibitor. In particular, we observed a higher mortality rate concomitant with an increase in the knockdown phenotype. Starvation prior to blood feeding also increased the effects of L-cycloserine with a rapid increase in mortality. CONCLUSIONS: Our results provide evidence that exposure of high doses of L-cycloserine during A. aegypti blood feeding affects mosquito survival and motor activity, suggesting an interference with carbohydrate and ammonia metabolism in a time-dependent manner.
Assuntos
Aedes/efeitos dos fármacos , Antimetabólitos/farmacologia , Comportamento Animal/efeitos dos fármacos , Ciclosserina/farmacologia , Aedes/fisiologia , Alanina Transaminase/metabolismo , Animais , Feminino , Glucose/farmacologia , Controle de Mosquitos , Atividade Motora/efeitos dos fármacos , SacaroseRESUMO
Aedes aegypti mosquitoes do not have a typical functional urea cycle for ammonia disposal such as the one present in most terrestrial vertebrates. However, they can synthesize urea by two different pathways, argininolysis and uricolysis. We investigated how formation of urea by these two pathways is regulated in females of A. aegypti. The expression of arginase (AR) and urate oxidase (UO), either separately or simultaneously (ARUO) was silenced by RNAi. The amounts of several nitrogen compounds were quantified in excreta using mass spectrometry. Injection of mosquitoes with either dsRNA-AR or dsRNA-UO significantly decreased the expressions of AR or UO in the fat body (FB) and Malpighian tubules (MT). Surprisingly, the expression level of AR was increased when UO was silenced and vice versa, suggesting a cross-talk regulation between pathways. In agreement with these data, the amount of urea measured 48 h after blood feeding remained unchanged in those mosquitoes injected with dsRNA-AR or dsRNA-UO. However, allantoin significantly increased in the excreta of dsRNA-AR-injected females. The knockdown of ARUO mainly led to a decrease in urea and allantoin excretion, and an increase in arginine excretion. In addition, dsRNA-AR-injected mosquitoes treated with a specific nitric oxide synthase inhibitor showed an increase of UO expression in FB and MT and a significant increase in the excretion of nitrogen compounds. Interestingly, both a temporary delay in the digestion of a blood meal and a significant reduction in the expression of several genes involved in ammonia metabolism were observed in dsRNA-AR, UO or ARUO-injected females. These results reveal that urea synthesis and excretion in A. aegypti are tightly regulated by a unique cross-talk signaling mechanism. This process allows blood-fed mosquitoes to regulate the synthesis and/or excretion of nitrogen waste products, and avoid toxic effects that could result from a lethal concentration of ammonia in their tissues.
Assuntos
Aedes/metabolismo , Ureia/metabolismo , Amônia/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Corpo Adiposo/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Túbulos de Malpighi/enzimologia , Redes e Vias Metabólicas , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Interferência de RNA , Urato Oxidase/genética , Urato Oxidase/metabolismo , VitelogêneseRESUMO
Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS(2)) to monitor each carbon of the above isotopically-labeled (13)C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS(2) tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS(2) fragmentation pathways are proposed for them. It was also found that small fragments (≤m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify (13)C-amino acids labeled at various positions, either in the backbone or side chain.
Assuntos
Aminoácidos/química , Espectrometria de Massas em Tandem/métodos , Aminoácidos/metabolismo , Isótopos de Carbono/química , Metilação , Modelos Moleculares , Prótons , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
It has been demonstrated that argininolysis and uricolysis are involved in the synthesis and excretion of urea in Aedes aegypti female mosquitoes. To further investigate the metabolic regulation of urea in female mosquitoes, it is desirable to have a rapid and efficient method to monitor arginine (Arg) concentration in mosquito excreta. Thus, a procedure currently used for the identification of Arg in urea cycle disorders in newborn babies was adapted to analyze Arg in A. aegypti excreta. The fragmentation patterns of the isobutyl esters of Arg and (15)N(2)-Arg (labeled at the guanidino group) were explored by electrospray ionization (ESI)-tandem mass spectrometry and fragmentation pathways not described before were characterized. In addition, Arg, (18)O(2)-Arg, (15)N(2)-Arg and (15)N(2)-(18)O(2)-Arg were also analyzed to elucidate some of the minor fragments in greater detail. Mosquito excreta from individual females were collected before and at different times after feeding a blood meal, mixed with (15)N(2)-Arg, an internal standard, and then derivatized as isobutyl esters. Based on the fragmentation mechanisms of Arg standards, studied by MS(2) and MS(3), Arg in the mosquito excreta was successfully analyzed by ESI-multiple reaction monitoring in a triple-quadrupole mass spectrometer. Arg excretion was monitored over a 120 h window before and after feeding female mosquitoes with a blood meal, with the maximum level of Arg excretion observed at 36-48 h post blood feeding. This method provides an efficient and rapid tool to quantify Arg in individual blood-fed mosquitoes, and can be applied to other organisms, whose small size severally limits the use of conventional biochemical analysis.
Assuntos
Aedes/metabolismo , Arginina/análogos & derivados , Arginina/análise , Fezes/química , Animais , Arginina/química , Ésteres/análise , Ésteres/química , Feminino , Humanos , Prolina/química , Prolina/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Ureia/metabolismoRESUMO
The fragmentation patterns of various (13)C-labeled glucose molecules were analyzed by electrospray ionization tandem mass spectrometry. Derivatization of glucose to yield methylglucosamine makes the C-C bond between C1 and C2 a favored cleavage site. This is in contrast to underivatized glucose, which favorably undergoes loss of a fragment containing both C1 and C2. Based on the fragmentation pattern of methylglucoasmine, we developed a method to distinguish and quantify C1 and C2 (13)C-labeled glucose by derivatization with methylamine followed by multiple reaction monitoring scans in a Q-trap mass spectrometer. Fragment ion ratios in the tandem mass spectra showed an isotope effect with (13)C or deuterium labeling, so a "correction factor" was introduced to make the quantification more accurate. The current approach can be applied to individually monitor the metabolic origin and fate of C1 and C2 atoms in (13)C-labeled glucose. This method provides a new means of quantifying glucose isotopomers in metabolic studies.
Assuntos
Glucose/análise , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/química , Deutério/química , Glucose/química , Marcação por IsótopoRESUMO
In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of (15)N from (15)NH(4)Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and (15)NH(4)Cl for 10-40min. The media were then mixed with deuterium-labeled amino acids, dried and derivatized. The (15)N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the (15)N from (15)NH(4)Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, (15)N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes.
Assuntos
Aedes/metabolismo , Amônia/metabolismo , Corpo Adiposo/metabolismo , Trato Gastrointestinal/metabolismo , Redes e Vias Metabólicas/fisiologia , Alanina Transaminase/metabolismo , Aminoácidos/metabolismo , Animais , Feminino , Glutamato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Técnicas In Vitro , Espectrometria de Massas , Isótopos de Nitrogênio/metabolismoRESUMO
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.
Assuntos
Aedes/metabolismo , Regulação da Expressão Gênica , Ureia/metabolismo , Alantoína/química , Animais , Feminino , Glutamina/química , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Nanotecnologia/métodos , Nitrogênio/química , Interferência de RNA , Espectrometria de Massas por Ionização por Electrospray , Urato Oxidase/metabolismo , Ureia/análogos & derivados , Ureia/química , Ácido Úrico/químicaRESUMO
We have established a protocol to study the kinetics of incorporation of 15N into glutamine (Gln), glutamic acid (Glu), alanine (Ala) and proline (Pro) in Aedes aegypti females. Mosquitoes were fed 3% sucrose solutions containing either 80 mM 15NH4Cl or 80 mM glutamine labeled with 15N in either the amide nitrogen or in both amide and amine nitrogens. In some experiments, specific inhibitors of glutamine synthetase or glutamate synthase were added to the feeding solutions. At different times post feeding, which varied between 0 and 96 h, the mosquitoes were immersed in liquid nitrogen and then processed. These samples plus deuterium labeled internal standards were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. The quantification of 15N-labeled and unlabeled amino acids was performed by using mass spectrometry techniques. The results indicated that the rate of incorporation of 15N into amino acids was rapid and that the label first appeared in the amide side chain of Gln and then in the amino group of Gln, Glu, Ala and Pro. The addition of inhibitors of key enzymes related to the ammonia metabolism confirmed that mosquitoes efficiently metabolize ammonia through a metabolic route that mainly involves glutamine synthetase (GS) and glutamate synthase (GltS). Moreover, a complete deduced amino acid sequence for GltS of Ae. aegypti was determined. The sequence analysis revealed that mosquito glutamate synthase belongs to the category of NADH-dependent GltS.
Assuntos
Aedes/metabolismo , Amônia/metabolismo , Glutamato Sintase/metabolismo , Aedes/enzimologia , Sequência de Aminoácidos , Aminoácidos/biossíntese , Animais , Glutamato Sintase/química , Glutamina/metabolismo , Cinética , Dados de Sequência Molecular , Radioisótopos de Nitrogênio/metabolismoRESUMO
A fragmentation mechanism for the neutral loss of 73 Da from dimethylformamidine glutamine isobutyl ester is investigated. Understanding this mechanism will allow to improve the identification and quantification of 15N-labeled and unlabeled glutamine and the distinguishing of glutamine and glutamic acid by electrospray ionization (ESI)-tandem mass spectrometry. Before mass spectrometry analysis, glutamine and glutamic acid are derivatized with dimethylformamide dimethyl acetal and isobutanol to form dimethylformamidine isobutyl ester. Derivatization conditions are modified based on an existing method to ensure complete derivatization of glutamic acid and to prevent the hydrolysis of glutamine. The fragmentation mechanism of dimethylformamidine glutamine isobutyl ester is studied and possible fragmentation pathways are proposed. Based on the fragmentation mechanism, a quantification method is developed to quantify both 15N-labeled and unlabeled glutamine and glutamic acid at a series of different neutral losses by performing multiple-reaction monitoring (MRM) scans in a triple-quadrupole mass spectrometer. Labeled glutamine includes 15N-amide labeled, 15N-amine labeled glutamine and glutamine 15N-labeled at both amide and amine positions. Deuterium labeled glutamine and glutamic acid are used as internal standards. Isotope effects are characterized for 15N labeled and deuterium labeled glutamine. It is found that the same method can be used to distinguish aspartic acid from asparagine. This study will improve the application of MS/MS for amino acid quantification and stable isotope labeling metabolism studies.