Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues

Abstract

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 10-40 minutes. The media was 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. Keywordslabeled amino acids; metabolic pathways; mass spectrometry; nitrogen compounds 1-INTRODUCTIONAedes aegypti mosquitoes are vectors of medical importance. During blood feeding, A. aegypti females are able to transmit viruses that produce serious infectious diseases such as Dengue fever and Yellow fever causing significant morbidity and mortality worldwide. In recent years, the A. aegypti populations have been spread significantly increasing the risk of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Certainly, A. aegypti females are a remarkable biological system. In spite of the vast amount of blood meal proteins that females ingest in a short time, and the tremendous amino acid oxidation that occurs during blood metabolism (Zhou et al., 2004), A. aegypti females are able to utilize dietary nutrients and efficiently remove toxic compounds and excess nitrogen. It is of interest to uncover the metabolic mechanisms that A. aegypti female mosquitoes use to avoid toxic accumulation of ammonia in the tissues, which result from massive amino acid deamination, in order to potentially develop new strategies for vector control. In this article, the term "ammonia" refers to...

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti

“…S6A), a protein known to control the juvenile-to-adult transition in C. elegans (49). Another protein, GS1 (AAEL001887, glutamine synthetase 1), involved in ammonia detoxification, is critical in both FB and midgut (50) and was found to be potentially targeted by miR-2940-5p (Dataset S4). Thus, it will be interesting to further examine the effects of miR-275-3p and miR-2940-5p on nitrogen metabolism.…”

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

“…The Western blot shown is representative of 3 replicates. midgut were incubated with 15 N-labeled ammonium salts, indicating the functional importance of ALAT during an ammonia challenge (8).…”

“…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).…”

Aphid genome expression reveals host–symbiont cooperation in the production of amino acids

“…Among other insects, the GS/GltS cycle has been characterized in vivo in mosquitoes (31,32) and silkworms (33)(34)(35). In the silkworm, GltS activity is associated with nitrogen recycling through the GS/GltS cycle; GltS activation in the posterior silk glands allows enhanced utilization of nitrogen (in the form of glutamate converted from glutamine) for the synthesis of silk protein (33)(34)(35).…”