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

Abstract

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

Xanthine dehydrogenase‐1 silencing in Aedes aegypti mosquitoes promotes a blood feeding–induced adulticidal activity

“…aegypti females with a deficiency in arginase or/and urate oxidase (19). In contrast, XDH1 mRNA abundance increases in fat body, thorax, and malpighian tubules from mosquitoes with disrupted ALT expression (21). To investigate the expression pattern of genes encoding XDH in wild‐type mosquitoes, we analyzed both XDH1 and XDH2 mRNA abundance by real‐time qPCR in several tissues dissected before and after blood feeding.…”

“…Mosquito excreta were collected at 24, 36, 48, and 72 h post–blood meal (PBM), whereas malpighian tubules were dissected without a buffer solution at 6, 12, 24, 36, 48, 72, and 96 h PBM. Uric acid and heme concentrations were quantified as previously described (21) with a minor modification for the heme assay: samples were completely dried using an Eppendorf Vacufuge plus concentrator (Hauppauge, NY, USA) and reconstituted with 33 μl of 0.1% KOH.…”

“…gambiae and D. melanogaster , respectively, suggesting that XDH2 arose from a gene duplication in the lineage leading to the Aedes genus. The methods utilized for the synthesis of double‐stranded RNA (dsRNA) against XDH1, XDH2, and firefly luciferase (FL) control, dsRNA microinjection and real‐time qPCR analysis have been previously described (19, 21). The relative mRNA levels for XDH1, XDH2, ammonia transporter (Rh50–1), glutamine synthetase (GS1 and GS2), ALT1, ALT2, NMDA receptor (NMDAR), and antioxidant genes including catalase (CAT1 and CAT2), thioredoxin reductase (TRX), superoxide dismutase (SOD1 and SOD2), and ornithine decarboxylase (ODC) in response to XDH knockdown were measured in mosquito tissues at 24 h PBM.…”

“…aegypti survival (20), whereas reduced levels of ALT1 or ALT2 by RNA interference (RNAi) slightly impair motor activity without affecting mosquito survival. Silencing of ALT also causes a massive but temporary increase of uric acid in the midgut and a delay in digestion, excretion, and oviposition with a significant reduction in egg production (21). Additionally, knockdown of ALT1 or ALT2 causes a concomitant increase in the transcript levels of both the ammonia transporter Rhesus 50 glycoprotein (Rh50–1) and XDH1 , possibly to avoid cell toxicity.…”

How micronutrients influence the physiology of mosquitoes

“…Proline again occupies a central role in nitrogen metabolism and ammonia detoxification by serving as a nitrogen sink during the rapid deamination of proteins and production of ammonia that occurs following a blood meal [25]. Preventing ammonia detoxification by RNAi knockdown of ALAT, causes a massive increase of uric acid in the midgut and a delay in digestion, excretion, and oviposition with a significant reduction in egg production [26]. Treatment with the ALAT inhibitor L-cycloserine resulted in significant increases in mortality and disruptions to motor activity in a dose-dependent manner [27].…”