Toxicity of Ulva lactuca and green fabricated silver nanoparticles against mosquito vectors and their impact on the genomic DNA of the dengue vector Aedes aegypti.

Marine seaweeds are known to have a potential role against microbial and pesticidal activities. Ulva lactuca, a green macroalgae extract analysed through gas chromatography mass spectrometry reveals 31 compounds. Resistance of mosquito vectors to synthetic insecticides remains a major problem. Discovering and applying natural agents to act against disease vectors is challenging. The activities of the extract and nano-fabricated green synthesised silver nanoparticles were checked for use against Aedes aegypti and Culex pipiens. The crude extract and synthesised silver nanoparticles exhibited a notable larvicidal effect, and very effective inhibition of pupal and adult emergence. Inhibition of adult emergence of Ae.aegypti was 97.7% and in Cu.pipiens, it was 93.3%. Our genotypic study of Deoxyribonucleic acid from treated larvae utilising random primers MA-09, MA-12 and MA-26 revealed damaged nucleotide sequences when compared with the controls. The antimicrobial activity of both the extract and green synthesised nanomaterials showed prominent activity against pathogenic drug resistant bacteria. Our results contribute to further development of eco-friendly insecticides with lower cost of preparation. This could further contribute to further research helping future generations to be free from these deadly disease-causing vectors and pathogenic microbes.

Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions.

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies.

Mosquitoes (Diptera: Culicidae) serve as important vectors for a wide number of parasites and pathogens of huge medical and veterinary importance. Aedes aegypti is a primary dengue vector in tropical and subtropical urban areas. There is an urgent need to develop eco-friendly mosquitocides. In this study, silver nanoparticles (AgNP) were biosynthesized using neem cake, a by-product of the neem oil extraction from the seed kernels of Azadirachta indica. AgNP were characterized using a variety of biophysical methods, including UV-vis spectrophotometry, FTIR, SEM, EDX, and XRD analyses. Furthermore, the neem cake extract and the biosynthesized AgNP were tested for acute toxicity against larvae and pupae of the dengue vector Ae. aegypti. LC50 values achieved by the neem cake extract ranged from 106.53 (larva I) to 235.36 ppm (pupa), while AgNP LC50 ranged from 3.969 (larva I) to 8.308 ppm (pupa). In standard laboratory conditions, the predation efficiency of a Carassius auratus per day was 7.9 (larva II) and 5.5 individuals (larva III). Post-treatment with sub-lethal doses of AgNP, the predation efficiency was boosted to 9.2 (larva II) and 8.1 individuals (larva III). The genotoxic effect of AgNP was studied on C. auratus using the comet assay and micronucleus frequency test. DNA damage was evaluated on peripheral erythrocytes sampled at different time intervals from the treatment; experiments showed no significant damages at doses below 12 ppm. Overall, this research pointed out that neem cake-fabricated AgNP are easy to produce, stable over time, and can be employed at low dosages to reduce populations of dengue vectors, with moderate detrimental effects on non-target mosquito natural enemies.

Effect of isodillapiole on the expression of the insecticide resistance genes GSTE7 and CYP6N12 in Aedes aegypti from central Amazonia.

The yellow fever mosquito Aedes (Stegomyia) aegypti is the main vector of dengue arbovirus and other arboviruses. Dengue prevention measures for the control of A. aegypti involve mainly the use of synthetic insecticides. The constant use of insecticides has caused resistance in this mosquito. Alternative studies on plant extracts and their products have been conducted with the aim of controlling the spread of the mosquito. Dillapiole is a compound found in essential oils of the plant Piper aduncum (Piperaceae) which has been effective as a biopesticide against A. aegypti. Isodillapiole is a semisynthetic substance obtained by the isomerization of dillapiole. In the present study, isodillapiole was evaluated for its potential to induce differential expression of insecticide resistance genes (GSTE7 and CYP6N12) in 3rd instar larvae of A. aegypti. These larvae were exposed to this compound at two concentrations (20 and 40 µg/mL) for 4 h during four generations (G1, G2, G3, and G4). Quantitative RT-PCR was used to assess the expression of GSTE7 and CYP6N12 genes. GSTE7 and CYP6N12 relative expression levels were higher at 20 than at 40 µg/mL and varied among generations. The decrease in GSTE7 and CYP6N12 expression levels at the highest isodillapiole concentration suggests that larvae may have suffered from metabolic stress, revealing a potential alternative product in the control of A. aegypti.

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti.

Inward-rectifying K(+) (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K(+) currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na(+). Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl(+) > K(+) > Rb(+) > NH(4)(+)) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K(+).

Molecular cloning and characterization of the complete acetylcholinesterase gene (Ace1) from the mosquito Aedes aegypti with implications for comparative genome analysis.

Insensitive acetylcholinesterase (AChE) has been shown to be responsible for resistance to organophosphates and carbamates in a number of arthropod species. Some arthropod genomes contain a single Ace gene, while others including mosquitoes contain two genes, but only one confers insecticide resistance. Here we report the isolation of the full-length cDNA and characterization of the complete genomic DNA sequence for the Ace1 gene in the yellow fever mosquito, Aedes aegypti. The Ace1 homolog in other mosquito species has been associated with insecticide resistance. The full-length cDNA consists of 2721bp and contains a 2109bp open reading frame that encodes a 702 amino acid protein. The amino acid sequence is highly conserved with that of other mosquitoes, including greater than 90% identity with Culex spp. and about 80% identity with Anopheles gambiae. The genomic DNA sequence includes 138,970bp and consists of eight exons with seven introns ranging from 59 to 114,350bp. Exons 2 and 8 show reduced amino acid conservation across mosquito species, while exons 3-7 are highly conserved. The Ace1 introns in Ae. aegypti reflect a high frequency of repetitive sequences that comprise about 45% of the total intron sequence. The Ace1 locus maps to the p-arm of chromosome 3, which corresponds to the orthologous genome regions in Culex spp. and An. gambiae.

Identification of two sterol carrier protein-2 like genes in the yellow fever mosquito, Aedes aegypti.

Two genes encoding sterol carrier protein-2 like proteins are identified from fourth instar cDNAs of the yellow fever mosquito, Aedes aegypti. The predicted AeSCP-2like1 (AeSCP-2L1) and AeSCP-2like2 (AeSCP-2L2) proteins are small, acidic and lacking the peroxisomal targeting sequence at the C-termini. Purified recombinant AeSCP-2L1 and -2L2 bind to cholesterol with a Kd of 5.4 x 10(-6) M and 2.6 x 10(-6) M, respectively. The Kd values of AeSCP-2L1 and -2L2 to palmitic acid are 3.7 x 10(-7) M and 2.6 x 10(-7) M, respectively. Both genes are expressed predominantly in gut tissues. The transcripts of the AeSCP-2L1 gene are only detected in larval stages, whereas AeSCP-2L2 is expressed in larval and adult stages. AeSCP-2L2 transcription increases within 5 h after a bloodmeal and stays at high levels during vitellogenesis. In in vitro larval gut tissue cultures, AeSCP-2L1 transcripts were increased in the presence of juvenile hormone III, whereas AeSCP-2L2 mRNA levels increased in the presence 20-hydroxylecdysone. The results suggest that transcription of AeSCP-2L1 and -2L2 genes are regulated differently through the mosquito life cycle.

Cloning and characterization of cDNAs encoding putative CTCFs in the mosquitoes, Aedes aegypti and Anopheles gambiae.

BACKGROUND: One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes. RESULTS: We have identified and characterized putative CTCF homologs in the medically-important mosquitoes, Aedes aegypti and Anopheles gambiae. These genes encode polypeptides with eleven C2H2 zinc fingers that show significant similarity to those of vertebrate CTCFs, despite at least 500 million years of divergence. The mosquito CTCFs are constitutively expressed and are upregulated in early embryos and in the ovaries of blood-fed females. We have uncovered significant bioinformatics evidence that CTCF is widespread, at least among Drosophila species. Finally, we show that the An. gambiae CTCF binds two known insulator sequences. CONCLUSION: Mosquito CTCFs are likely orthologous to the widely-characterized vertebrate CTCFs and potentially also serve an insulating function. As such, CTCF may provide a powerful tool for improving transgene expression in these mosquitoes through the identification of endogenous binding sites.

Mosquito glucosamine-6-phosphate N-acetyltransferase: cDNA, gene structure and enzyme kinetics.

Mosquito midgut epithelial cells secrete digestive enzymes as well as components of the peritrophic matrix in response to blood-feeding. The peritrophic matrix is composed of proteins, glycoproteins and chitin fibrils in a proteoglycan matrix and may function to protect the midgut epithelium from mechanical damage and insult from pathogens and toxins. Chitin biosynthesis takes place via the hexosamine pathway converting fructose-6-phosphate to UDP-N-acetylglucosamine, which is then polymerized to chitin by chitin synthase. Glucosamine-6-phosphate N-acetyltransferase (GNA) is one of the hexosamine pathway enzymes and catalyzes the transfer of the acetyl group from acetyl-CoA to the primary amine of glucosamine-6-phosphate. We cloned and sequenced the GNA cDNA, gene (AeGna) and its putative promoter regions from Aedes aegypti. AeGna consists of five exons and four introns and lacks a TATA box near the transcription start site. The AeGna cDNA is 1.3 kb in length and the predicted protein is approximately 23.6 kDa. The amino acid sequence of AeGna has high homology to its orthologues. AeGna mRNA is constitutively expressed in all developmental stages and blood-feeding causes no obvious effect on levels of AeGna transcript in the midgut. The Km value of recombinant GNA for glucosamine-6-phosphate was 330 microM and the Km for acetyl-CoA was 500 microM.

Identification, functional characterization and expression of a LAT type amino acid transporter from the mosquito Aedes aegypti.

We isolated two cDNAs from the mosquito Aedes aegypti, an L-amino acid transporter (AeaLAT) and a CD98 heavy chain (AeaCD98hc). Expression of AeaCD98hc or AeaLAT alone in Xenopus oocyte did not induce amino acid transport activity. However, co-expression of AeaCD98hc and AeaLAT, which are postulated to form a heterodimer protein linked through a disulfide bond, showed significant increase in amino acid transport activity. This heterodimeric protein showed uptake specificity for large neutral and basic amino acids. Small acidic neutral amino acids were poor substrates for this transporter. Neutral amino acid (leucine) uptake activity was partially Na+ dependent, because leucine uptake was approximately 44% lower in the absence of Na+ than in its presence. However, basic amino acid (lysine) uptake activity was completely Na+ independent at pH of 7.4. Extracellular amino acid concentration could be the main factor that determined amino acid transport. These results suggest the heteromeric protein is likely a uniporter mediating diffusion of amino acids in the absence of ions. The AeaLAT showed high level expression in the gastric caeca, Malpighian tubules and hindgut of larvae. In caeca and hindgut expression was in the apical cell membrane. However, in Malpighian tubules and in midgut, the latter showing low level expression, the transporter was detected in the basolateral membrane. This expression profile supports the conclusion that this AeaLAT is a nutrient amino acid transporter.

Ribonucleotide reductase subunits from the yellow fever mosquito, Aedes aegypti: cloning and expression.

Ribonucleotide reductase catalyses the de novo synthesis of deoxyribonucleotides. Class I reductases use an iron center to generate a tyrosyl free radical that can initiate formation of the deoxyribonucleotide. These reductases are alpha 2 beta 2 holoenzymes, and the subunits are denoted as R1 and R2. R1 contains the allosteric binding site and the active site, whereas R2 contains a binuclear iron center that initiates formation of the tyrosyl radical. We have cloned and sequenced the cDNAs encoding the R1 and R2 subunit in the yellow fever mosquito, Aedes aegypti. The messages for these proteins are increased in response to blood-feeding.

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti.

Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP-N-acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5' regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

Aedes aegypti glutamine synthetase: expression and gene structure.

The peritrophic matrix (PM) is the first natural barrier a mosquito-borne parasite faces when ingested with a blood meal; consequently, understanding the biology of PM formation could provide novel transmission control strategies. Because the PM is composed of chitin (a molecule of repeating units of N-acetyl glucosamine), glycoproteins and glucose, characterizing the regulation of enzymes involved in chitin production should provide information concerning factors that influence PM formation. We previously have shown that glutamine synthetase (GS) provides the glutamine needed in the initial steps of chitin biosynthesis in the yellow fever mosquito, Aedes aegypti. In the present study we show that GS is encoded by a single 4.5 kb gene, designated mGS, containing three exons and two introns. Multiple transcripts are generated from mGS presumably by differential splicing of the introns. Sequences of two cDNAs encoding GS are identical at the protein level, but differ in their 5'-untranslated regions. GS message is constitutively expressed in all developmental stages and in most tissues, with an increase in GS transcription observed in midgut and fat body tissues of female mosquitoes following a blood meal. Transcripts are localized to the apical side of the mosquito midgut epithelium and data suggest that mGS transcription is regulated by an Oct-1 transcription factor.

Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

Molecular cloning of two prophenoloxidase genes from the mosquito Aedes aegypti.

The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro-PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full-length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro-PO. It contains two putative copper binding domains (amino acids 197-243 and 346-423) that are homologous to other insect pro-POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT-PCR) only from third-stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT-PCR from mRNA obtained from 14-day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar.

Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA.

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine-rich domains, two of which flank a proline/serine/threonine-rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal-responsive gene from an aquatic insect.

Aedes aegypti dopa decarboxylase: gene structure and regulation.

Dopa decarboxylase converts L-dopa to dopamine, a precursor molecule for diverse biological activities in insects including neurotransmission and a variety of tanning reactions required for development, reproduction and defence against parasites. Herein, we report the cloning and sequencing of the Aedes aegypti Ddc gene, including 2.1 kb of the upstream promoter region. The transcribed region of the gene spans more than 16 kb and contains five exons. In situ hybridization localizes the blood-meal-induced ovarian transcription of this gene to the follicular epithelial cells surrounding individual oocytes. Ovary tissue transcription of Ddc is increased in response to injection of 20-hydroxyecdysone to levels equal to those observed for blood-fed controls, however coinjection with the translational inhibitor cycloheximide negates the effect, indicating an indirect regulatory role for this hormone. Clusters of putative ecdysone-responsive elements and zinc-finger binding domains for the products of Broad-Complex gene family are identified in the 5'-promoter region. These elements are discussed in the context of common insect Ddc regulatory mechanisms.

A novel member of the RING-finger gene family associated with reproductive tissues of the mosquito, Aaedes aegypti.

The RING finger is a zinc-binding domain that is found in proteins from viruses, plants and animals. Here we report the characterization and tissue-specific expression of a mosquito gonadal protein gene, mgp, from the mosquito, Aedes aegypti. The putative gene product, MGP, contains two RING fingers, a B-box, and a hydrophobic core. These mosquito MGP structural motifs are highly conserved in proteins found in mouse and nematode. Northern blot analysis and in situ hybridization demonstrated the presence of multiple mgp RNA transcripts in male and female reproductive tissues. Expression of mgp in the ovary is constitutive, but an increase in message was observed in the ovaries of female mosquitoes previously exposed to a blood meal. These results suggest that MGP is a protein that might play a role(s) in mosquito gametogenesis.

Green fluorescent protein as a genetic marker in transgenic Aedes aegypti.

We report here the use of the enhanced green fluorescent protein (EGFP) from the jellyfish, Aequorea victoria, as a genetic marker for the genetic transformation of mosquitoes. The EGFP gene, under the control of the actin5C promoter of Drosophila melanogaster was inserted into the Hermes transposable element. Preblastoderm embryos of a wild-type strain of the yellow fever mosquito, Aedes aegypti, were microinjected with this plasmid, together with a helper plasmid containing the Hermes transposase placed under the control of the D. melanogaster hsp70 promoter. Somatic EGFP expression was observed during early instars in approximately one-half of all G0 individuals. Two G1 individuals arising from a G0 female displayed high levels of EGFP gene expression during all stages of development. EGFP was transmitted in a Mendelian fashion to the G2 and G3 generations and molecular analysis confirmed the presence of the Hermes[actin5C:EGFP] gene in these insects. These results clearly demonstrate that EGFP can be used as an effective genetic marker in wild-type Ae. aegypti and most likely in other mosquito species as well.

Biosynthesis of Aedes aegypti lipophorin and gene expression of its apolipoproteins.

The biosynthesis of lipophorin of the yellow fever mosquito, Aedes aegypti, was investigated. Fat bodies were incubated in vitro with radiolabeled methionine and cysteine, and radiolabeled proteins secreted into the medium were analyzed by density gradient ultracentrifugation, SDS-PAGE and fluorography. Lipophorin was synthesized in the fat body and secreted into the medium. Its density was 1.114 g/ml, similar to that of lipophorin circulating in hemolymph. Three peptides of a tryptic digest of apolipophorin II were sequenced and degenerate oligonucleotide primers were designed based on the amino acid sequences. With these primers, a cDNA product of 1.2 kb was amplified by RT-PCR using as template RNA extracted from adult female mosquitoes 24 h after ingestion of a blood meal. This cDNA was cloned, sequenced and used as a probe for Northern blot analysis, which revealed that the apoproteins of lipophorin were coded for by a single mRNA of approximately 10 kb. The expression of the apolipophorins was induced by blood feeding. From the data presented we concluded that Aedes aegypti lipophorin is synthesized in the fat body and that the expression of its apolipophorins is induced by blood feeding.