Characterization of essential eggshell proteins from Aedes aegypti mosquitoes.

BACKGROUND: Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti, coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. RESULTS: In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. CONCLUSIONS: While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies.

Ornithine decarboxylase deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti mosquitoes.

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.

Identification and characterization of a mosquito-specific eggshell organizing factor in Aedes aegypti mosquitoes.

Mosquito-borne diseases are responsible for several million human deaths annually around the world. One approach to controlling mosquito populations is to disrupt molecular processes or antagonize novel metabolic targets required for the production of viable eggs. To this end, we focused our efforts on identifying proteins required for completion of embryonic development that are mosquito selective and represent potential targets for vector control. We performed bioinformatic analyses to identify putative protein-coding sequences that are specific to mosquito genomes. Systematic RNA interference (RNAi) screening of 40 mosquito-specific genes was performed by injecting double-stranded RNA (dsRNA) into female Aedes aegypti mosquitoes. This experimental approach led to the identification of eggshell organizing factor 1 (EOF1, AAEL012336), which plays an essential role in the formation and melanization of the eggshell. Eggs deposited by EOF1-deficient mosquitoes have nonmelanized fragile eggshells, and all embryos are nonviable. Scanning electron microscopy (SEM) analysis identified that exochorionic eggshell structures are strongly affected in EOF1-deficient mosquitoes. EOF1 is a potential novel target, to our knowledge, for exploring the identification and development of mosquito-selective and biosafe small-molecule inhibitors.

Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

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.

Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase.

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.

Defects in coatomer protein I (COPI) transport cause blood feeding-induced mortality in Yellow Fever mosquitoes.

Blood feeding by vector mosquitoes provides the entry point for disease pathogens and presents an acute metabolic challenge that must be overcome to complete the gonotrophic cycle. Based on recent data showing that coatomer protein I (COPI) vesicle transport is involved in cellular processes beyond Golgi-endoplasmic reticulum retrograde protein trafficking, we disrupted COPI functions in the Yellow Fever mosquito Aedes aegypti to interfere with blood meal digestion. Surprisingly, we found that decreased expression of the γCOPI coatomer protein led to 89% mortality in blood-fed mosquitoes by 72 h postfeeding compared with 0% mortality in control dsRNA-injected blood-fed mosquitoes and 3% mortality in γCOPI dsRNA-injected sugar-fed mosquitoes. Similar results were obtained using dsRNA directed against five other COPI coatomer subunits (α, ß, ß', δ, and ζ). We also examined midgut tissues by EM, quantitated heme in fecal samples, and characterized feeding-induced protein expression in midgut, fat body, and ovary tissues of COPI-deficient mosquitoes. We found that COPI defects disrupt epithelial cell membrane integrity, stimulate premature blood meal excretion, and block induced expression of several midgut protease genes. To study the role of COPI transport in ovarian development, we injected γCOPI dsRNA after blood feeding and found that, although blood digestion was normal, follicles in these mosquitoes were significantly smaller by 48 h postinjection and lacked eggshell proteins. Together, these data show that COPI functions are critical to mosquito blood digestion and egg maturation, a finding that could also apply to other blood-feeding arthropod vectors.

Visualization of Apoptotic Ovarian Follicles during Aedes aegypti Mosquito Egg Maturation by Fluorescent Imaging Studies.

In insects, oocyte resorption (oosorption) or follicular atresia is one of the key physiological processes and evolutionary strategies used to optimize reproductive fitness. Mosquitoes are ideal model organisms for studying egg maturation in arthropods, as their follicle development is initiated only following the ingestion of a blood meal, followed by a carefully orchestrated series of hormonally regulated events leading to egg maturation. A cohort of approximately 100 follicles per mosquito ovary begin developing synchronously. However, a significant fraction of follicles ultimately undergo apoptosis and oosorption, especially when available resources from the blood meal are limited. Therefore, simple, rapid, and reliable techniques to accurately evaluate follicular atresia are necessary to understand mechanisms underlying follicle development in insects. This protocol describes how to detect apoptotic follicle cells within the Aedes aegypti mosquito ovaries using a commercially available fluorescent-labeled inhibitor of caspases (FLICA). Caspases are key players in animal apoptosis. In this assay, the FLICA reagent enters the intracellular compartment of follicles in dissected mosquito ovaries and covalently binds to active caspases. The bound reagent remains within the cell and its fluorescent signal can be observed by confocal microscopy. Although this method was specifically developed for visualizing apoptotic ovarian follicles during Ae. aegypti mosquito egg development, it should be applicable to other mosquito tissues that undergo caspase-mediated program cell death in a time-dependent manner.

Mosquito Egg Development and Eggshell Formation.

Anautogenous female mosquitoes, which ingest a blood meal from warm-blooded vertebrates to produce eggs, have become a valuable model organism for investigating signaling pathways and physiological processes that occur during egg development. Different molecular pathways tightly regulate the initiation of egg development and are governed by a balance among different insect hormones. Gravid (mature egg-carrying) females deposit fully developed eggs at the end of each gonotrophic cycle, which is defined as the time interval between the ingestion of a blood meal to oviposition. An intact eggshell protects the oocyte and embryo inside from external factors such as desiccation, physical damage, etc., and the various eggshell proteins are spatially and temporary deposited during oogenesis. Additionally, follicle resorption (oosorption) during blood meal-induced mosquito ovarian follicle development is an adapted physiological process that optimizes reproductive fitness. Mosquito oocytes grow and mature synchronously throughout oogenesis; however, during the later stages of oogenesis, some oocytes may undergo oosorption if sufficient nutrients are unavailable. This introduction highlights how mosquito egg development can be used to investigate follicular resorption and identify proteins involved in eggshell formation in Aedes aegypti mosquitoes.

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

Pyruvate kinase is post-translationally regulated by sirtuin 2 in Aedes aegypti mosquitoes.

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.

Distinct biological effects of golgicide a derivatives on larval and adult mosquitoes.

A collection of Golgicide A (GCA) analogs has been synthesized and evaluated in larval and adult mosquito assays. Commercially available GCA is a mixture of four compounds. One enantiomer (GCA-2) of the major diastereomer in this mixture was shown to be responsible for the unique activity of GCA. Structure-activity studies (SAR) of the GCA architecture suggested that the pyridine ring was most easily manipulated without loss or gain in new activity. Eighteen GCA analogs were synthesized of which five displayed distinct behavior between larval and adult mosquitos, resulting in complete mortality of both Aedes aegypti and Anopheles stephensi larvae. Two analogs from the collection were shown to be distinct from the rest in displaying high selectivity and efficiency in killing An. stephensi larvae.

Manipulation of pantothenate kinase in Anopheles stephensi suppresses pantothenate levels with minimal impacts on mosquito fitness.

Pantothenate (Pan) is an essential nutrient required by both the mosquito vector and malaria parasite. We previously demonstrated that increasing pantothenate kinase (PanK) activity and co-enzyme A (CoA) biosynthesis led to significantly decreased parasite infection prevalence and intensity in the malaria mosquito Anopheles stephensi. In this study, we demonstrate that Pan stores in A. stephensi are a limited resource and that manipulation of PanK levels or activity, via small molecule modulators of PanK or transgenic mosquitoes, leads to the conversion of Pan to CoA and an overall reduction in Pan levels with minimal to no effects on mosquito fitness. Transgenic A. stephensi lines with repressed insulin signaling due to PTEN overexpression or repressed c-Jun N-terminal kinase (JNK) signaling due to MAPK phosphatase 4 (MKP4) overexpression exhibited enhanced PanK levels and significant reductions in Pan relative to non-transgenic controls, with the PTEN line also exhibiting significantly increased CoA levels. Provisioning of the PTEN line with the small molecule PanK modulator PZ-2891 increased CoA levels while provisioning Compound 7 decreased CoA levels, affirming chemical manipulation of mosquito PanK. We assessed effects of these small molecules on A. stephensi lifespan, reproduction and metabolism under optimized laboratory conditions. PZ-2891 and Compound 7 had no impact on A. stephensi survival when delivered via bloodmeal throughout mosquito lifespan. Further, PZ-2891 provisioning had no impact on egg production over the first two reproductive cycles. Finally, PanK manipulation with small molecules was associated with minimal impacts on nutritional stores in A. stephensi mosquitoes under optimized rearing conditions. Together with our previous data demonstrating that PanK activation was associated with significantly increased A. stephensi resistance to Plasmodium falciparum infection, the studies herein demonstrate a lack of fitness costs of mosquito Pan depletion as a basis for a feasible, novel strategy to control parasite infection of anopheline mosquitoes.

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti.

BACKGROUND: The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. RESULTS: We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k(cat)/K(M)) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin. CONCLUSIONS: These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes.

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.

Activation of Anopheles stephensi Pantothenate Kinase and Coenzyme A Biosynthesis Reduces Infection with Diverse Plasmodium Species in the Mosquito Host.

Malaria parasites require pantothenate from both human and mosquito hosts to synthesize coenzyme A (CoA). Specifically, mosquito-stage parasites cannot synthesize pantothenate de novo or take up preformed CoA from the mosquito host, making it essential for the parasite to obtain pantothenate from mosquito stores. This makes pantothenate utilization an attractive target for controlling sexual stage malaria parasites in the mosquito. CoA is synthesized from pantothenate in a multi-step pathway initiated by the enzyme pantothenate kinase (PanK). In this work, we manipulated A. stephensi PanK activity and assessed the impact of mosquito PanK activity on the development of two malaria parasite species with distinct genetics and life cycles: the human parasite Plasmodium falciparum and the mouse parasite Plasmodium yoelii yoelii 17XNL. We identified two putative A. stephensi PanK isoforms encoded by a single gene and expressed in the mosquito midgut. Using both RNAi and small molecules with reported activity against human PanK, we confirmed that A. stephensi PanK manipulation was associated with corresponding changes in midgut CoA levels. Based on these findings, we used two small molecule modulators of human PanK activity (PZ-2891, compound 7) at reported and ten-fold EC50 doses to examine the effects of manipulating A. stephensi PanK on malaria parasite infection success. Our data showed that oral provisioning of 1.3 nM and 13 nM PZ-2891 increased midgut CoA levels and significantly decreased infection success for both Plasmodium species. In contrast, oral provisioning of 62 nM and 620 nM compound 7 decreased CoA levels and significantly increased infection success for both Plasmodium species. This work establishes the A. stephensi CoA biosynthesis pathway as a potential target for broadly blocking malaria parasite development in anopheline hosts. We envision this strategy, with small molecule PanK modulators delivered to mosquitoes via attractive bait stations, working in concert with deployment of parasite-directed novel pantothenamide drugs to block parasite infection in the human host. In mosquitoes, depletion of pantothenate through manipulation to increase CoA biosynthesis is expected to negatively impact Plasmodium survival by starving the parasite of this essential nutrient. This has the potential to kill both wild type parasites and pantothenamide-resistant parasites that could develop under pantothenamide drug pressure if these compounds are used as future therapeutics for human malaria.

Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes.

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.

Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.

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 ∼57 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.

TOR signaling is required for amino acid stimulation of early trypsin protein synthesis in the midgut of Aedes aegypti mosquitoes.

Blood meal digestion in mosquitoes occurs in two phases, an early phase that is translationally regulated, and a late phase that is transcriptionally regulated. Early trypsin is a well-characterized serine endoprotease that is representative of other early phase proteases in the midgut that are only synthesized after feeding. Since the kinase Target of Rapamycin (TOR) has been implicated as a nutrient sensor in other systems, including the mosquito fat body, we tested if TOR signaling is involved in early trypsin protein synthesis in the mosquito midgut in response to feeding. We found that ingestion of an amino acid meal by female mosquitoes induces early trypsin protein synthesis, coincident with phosphorylation of two known TOR target proteins, p70S6 kinase (S6K) and the translational repressor 4E-Binding Protein (4E-BP). Moreover, in vitro culturing of midguts from unfed mosquitoes led to amino acid-dependent phosphorylation of S6K and 4E-BP which could be blocked by treatment with rapamycin, a TOR-specific inhibitor. Lastly, by injecting mosquitoes with TOR double stranded RNA (dsRNA) or rapamycin, we demonstrated that TOR signaling was required in vivo for both phosphorylation of S6K and 4E-BP in the midgut, and for translation of early trypsin mRNA in response to amino acid feeding. It may be possible to target the TOR signaling pathway in the midgut to inhibit blood meal digestion, and thereby, decrease fecundity and the spread of mosquito borne diseases.

Mosquito vitellogenin genes: Comparative sequence analysis, gene duplication, and the role of rare synonymous codon usage in regulating expression.

Comparative sequence analysis of mosquito vitellogenin (Vg) genes was carried out to gain a better understanding of their evolution. The genomic clones of vitellogenin genes were isolated and sequenced from all three subfamilies of the family Culicidae including Culicinae (Aedes aegypti, Ochlerotatus atropalpus, Ae. polynesiensis, Ae. albopictus, Ochlerotatus triseriatus and Culex quinquefasciatus), Toxorhynchitinae (Toxorhynchites amboinensis), and Anophelinae (Anopheles albimanus). Genomic clones of vitellogenin genes Vg-B and Vg-C were isolated from Ae. aegypti and sequenced. A comparison of Vg-B and Vg-C, with the previously characterized vitellogenin gene, Vg-A1, suggests that Vg-A1 and Vg-B probably arose by a recent gene duplication, and Vg-C apparently diverged from the two other members of the gene family in an earlier gene duplication event. Two vitellogenin genes orthologous to Vg-C were cloned from a Cx. quinquefasciatus DNA library, one of which is truncated at the N-terminal end. Single vitellogenin genes, orthologous to Vg-C, were cloned from the An. albimanus and Tx. amboinensis libraries. Incomplete sequences orthologous to Vg-B and Vg-C were isolated from the Oc. atropalpus library. Only partial sequences were isolated from Ae. polynesiensis, Ae. albopictus and Oc. triseriatus. Inferred phylogenetic relationships based on analysis of these sequences suggest that Vg-C was the ancestral gene and that a recent gene duplication gave rise to Vg-A1 and Vg-B after the separation of the genus Aedes. The deduced amino acid composition of mosquito vitellogenin proteins exhibits higher tyrosine and phenylalanine composition than other mosquito proteins except for the hexamerin storage proteins. Analysis of vitellogenin coding sequences showed that a majority of amino acid substitutions were due to conserved and moderately conserved changes suggesting that the vitellogenins are under moderately selective constrains to maintain tertiary structure. The vitellogenin genes of the three anautogenous mosquitoes, that require a blood meal to develop eggs, had very high synonymous codon usage biases similar to highly expressed genes of other organisms. On the other hand, the vitellogenin genes of autogenous mosquitoes, that develop at least one batch of eggs without a blood meal, exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the vitellogenin genes, where a high number of rarely used synonymous codons are present. It is hypothesized that rare synonymous codons have selectively accumulated in the signal peptide region to down-regulate the rate of translation initiation in the absence of a blood meal. Real-time PCR gene expression experiments showed that all three Ae. aegypti vitellogenin genes were highly expressed after a blood meal, and expressed in non-blood-fed females, males, larvae and pupae at trace levels. Sequences were deposited in GenBank (accession numbers: Ae. aegypti Vg-B, AY380797, Vg-C, AY373377; Oc. atropalpus Vg-B, AY691321, Vg-C, AY691322; Ae. polynesiensis Vg-A1, AY691318, Vg-B, AY691319, Vg-C, AY691320; Ae. albopictus Vg-A1, AY691316, Vg-C, AY691317; Oc. triseriatus Vg-C, AY691323; Cx. quinquefasciatus Vg-C1, AY691324, Vg-C2, AY691325; Tx. amboinensis Vg-C, AY691326; An. albimanus Vg-C, AY691327).

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

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.