Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

Scaraffia, Patricia Y.; Zhang, Qingfen; Wysocki, Vicki H.; Isoe, Jun; Wells, Michael A.

doi:10.1016/j.ibmb.2006.05.003

Cited by 38 publications

(70 citation statements)

References 30 publications

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“…It was also demonstrated that ALAT is involved in ammonia metabolism in blood-fed mosquitoes (4). When mosquitoes were provided with 3% sucrose supplemented with 15 N-labeled ammonium salts, it was shown that ALAT transfers the 15 N-amino group from 15 N-Glu into pyruvate and produces 15 N-Ala and a-ketoglutarate in mosquito whole body (6). Because ALAT catalyzes a reversible reaction, the enzyme can also produce 15 N-Glu and pyruvate by transferring back the 15 N-amino group from 15 N-Ala to a-ketoglutarate.…”

Section: Discussionmentioning

confidence: 99%

“…Fat body, thorax, Malpighian tubules, ovary, and midgut were dissected in 1X PBS from wild-type sugar-fed (SF) and blood-fed female mosquitoes at 3,6,12,18,24,30,36, and 48 h after blood feeding. Tissues were homogenized in 13 lysis buffer (13 PBS, 1.0% Triton X-100, 12 mM sodium deoxycholate, and 2% SDS), and the protein extracts were subjected to 12% SDS-PAGE separation and Western blot analysis as previously described (10).…”

Section: Protein Extraction and Western Blotting Proceduresmentioning

confidence: 99%

“…Next, we collected excreta at several time points during blood meal digestion (6,12,18,24,36, and 48 h after blood feeding) and measured the concentration of uric acid. We also measured the heme concentration from the same excreta, because the amount of heme defecated is an indication of the progress of blood meal digestion (24,25).…”

Section: Knockdown Of Alat Expression Induces Accumulation Of Uric Acmentioning

confidence: 99%

“…The term ammonia is defined here as NH 3 or NH 4 + or a combination of both. The application of isotopically labeled compounds and mass spectrometry, as well as RNA interference (RNAi), has demonstrated that A. aegypti females efficiently detoxify ammonia through 3 phases: fixation, assimilation, and excretion (5)(6)(7)(8)(9)(10). During this complex metabolic challenge, female mosquitoes synthesize specific amino acids, such as Gln, Glu, Ala, and Pro, through reactions catalyzed by several enzymes, including glutamine synthetase, glutamate synthase, glutamate dehydrogenase, pyrroline-5-carboxylate synthase, pyrroline-5-carboxylaye reductase, and alanine aminotransferase (ALAT).…”

mentioning

confidence: 99%

“…Mosquitoes also synthesize and excrete other nitrogen-containing compounds, such as uric acid, allantoin, allantoic acid, and urea. A. aegypti females accomplish this process by simultaneously using multiple metabolic pathways (4,6,7). It was recently demonstrated that urea originating through argininolysis and uricolysis is tightly regulated by a unique crosstalk signaling mechanism.…”

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confidence: 99%

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Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

Mazzalupo¹,

Isoe²,

Belloni

et al. 2015

FASEB j.

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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. FASEB J. 30, 111-120 (2016). www.fasebj.org

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Section: Discussionmentioning

confidence: 99%

Section: Protein Extraction and Western Blotting Proceduresmentioning

confidence: 99%

Section: Knockdown Of Alat Expression Induces Accumulation Of Uric Acmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

Mazzalupo¹,

Isoe²,

Belloni

et al. 2015

FASEB j.

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Comparative kinetics of fatty acid–amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

Lait

Lobaido

Wiester

et al. 2010

Arch Insect Biochem Physiol

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N-Linolenoyl-L-glutamine is one of several structurally similar fatty acid-amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane-associated alimentary tissue enzyme(s). FAC elicitors comprise 17-hydroxylated or non-hydroxylated linolenic acid coupled with L-glutamine or L-glutamate by an amide bond. We demonstrate in vitro biosynthesis of N-linolenoyl-L-glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N-linolenoyl-L-glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K(m) values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V(max) values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K(m) values for coupling of L-glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V(max) values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively.

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Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel)

Zhang

Wei

et al. 2018

Arch Insect Biochem Physiol

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In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.

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Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

Cited by 38 publications

References 30 publications

Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

Comparative kinetics of fatty acid–amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel)

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