Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

Anopheles (Nyssorhynchus) darlingi is an important malaria vector in South and Central America; however, little is known about molecular aspects of its biology. Genomic and proteomic analyses were performed on the salivary gland products of Anopheles darlingi. A total of 593 randomly selected, salivary gland-derived cDNAs were sequenced and assembled based on their similarities into 288 clusters. The putative translated proteins were classified into three categories: (S) secretory products, (H) housekeeping products and (U) products with unknown cell location and function. Ninety-three clusters encode putative secreted proteins and several of them, such as an anophelin, a thrombin inhibitor, apyrases and several new members of the D7 protein family, were identified as molecules involved in haematophagy. Sugar-feeding related enzymes (alpha-glucosidases and alpha-amylase) also were found among the secreted salivary products. Ninety-nine clusters encode housekeeping proteins associated with energy metabolism, protein synthesis, signal transduction and other cellular functions. Ninety-seven clusters encode proteins with no similarity with known proteins. Comparison of the sequence divergence of the S and H categories of proteins of An. darlingi and An. gambiae revealed that the salivary proteins are less conserved than the housekeeping proteins, and therefore are changing at a faster evolutionary rate. Tabular and supplementary material containing the cDNA sequences and annotations are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_darlingi_sialome/.

The Musca domestica larval hexamerin (MdHex-L) is a hexameric glycoprotein with an apparent native molecular weight of 500 kDa. Seven different cDNAs that encode MdHex-L subunits were cloned and sequenced. Furthermore, amino acid sequences of isolated subunits were determined by the Edman degradation method and compared to the conceptual translation products derived from the cloned cDNAs. The obtained data indicate the existence of multiple forms of MdHex-L subunits and that these multiple forms may be grouped into three categories according to their percentages of nucleotide sequence identity.

The salivary gland proteins of adult female Anopheles darlingi were fractionated by reverse-phase HPLC and the five major peaks were submitted for amino-terminal sequencing using automated Edman degradation. The amino acid sequence of one of the purified salivary gland proteins showed similarity with the D7r3 protein of An. gambiae. Cloning and sequencing of two cDNAs allowed the prediction of the complete sequence of the An. darlingi D7 protein. The D7r3 protein is present specifically in adult female salivary glands of An. darlingi and despite being one of the major salivary gland proteins its function is not known. Predictions of secondary and tertiary structures revealed the similarity of the An. darlingi D7 protein to insect odorant binding proteins. This suggests that D7 proteins may act as carriers of hydrophobic molecules in mosquito saliva.

Aedes aegypti larval hemolymph proteins were analyzed, and the major protein was characterized. The major protein, designated P1, is hexameric and is composed of subunits with molecular weights estimated to be 83,000. P1 is dissociated into its subunits when the pH is elevated from 7 to 9. This protein accumulates during the last larval instar and is not detected in adult mosquitoes. These characteristics, together with the high content of aromatic amino acids, include P1 in the arylphorin group of the insect storage proteins. Arch. Insect Biochem. Physiol. 34:191–201, 1997. © 1997 Wiley-Liss, Inc.

During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries. This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an Mr = 430 kDa, and subunits of Mr = 70 kDa. The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0. Two cDNA clones, F0 and F2, were isolated and analysed. The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP‐2 hexamerin. Recombinant protein from the F2‐cDNA is recognized by a specific anti‐NVFP serum. The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP. The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica .

Peroxynitrite, the potent oxidant formed by the fast reaction between nitric oxide and superoxide anion, has been suggested to be the reactive intermediate responsible for some of the pathologies associated with an over-production of nitric oxide, in this report, We demonstrate that both nitric oxide and peroxynitrite are formed during infection of the susceptible mouse strain, BALB/c, with Leishmania amazonensis. Nitric oxide was detected as the nitrosyl hemoglobin complex: by EPR analysis of blood drawn from mice at 35, 64 and 148 days of infection. The levels of nitrosyl hemoglobin complex increased with disease evolution, which in the murine model used is characterized by skin lesions, ulceration and visceralization of the parasites, Peroxynitrite formation was inferred from immunoreaction of homogenates obtained from footpad lesions in the late stages of the infection with anti-nitrotyrosine antibody; homogenates from parasites drawn from the lesions were also immunoreactive, although to a lesser extent. Analysis of protein homogenates by gel electrophoresis and western blots suggests that peroxynitrite may degrade proteins in vivo, in addition to nitrating them. The results demonstrate that peroxynitrite is formed during murine leishmaniasis and may play a role in the aggravation of the disease.

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 +/ 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage.

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).

The purified lipophorin of Aedes aegypti (Diptera) is composed of two apolipoproteins: apolipophorin I (M(r)=224,000) and apolipophorin II (M(r)=73,000). The density of lipophorin is constant during the Aedes life-cycle and equal to 1.11 +/- 0.01 g/ml. The amount of lipophorin per animal, during the gonotrophic cycles, increases until 48 hr after blood-feeding and then decreases until there is a new blood intake. The density values and quantification of lipophorin during Aedes aegypti gonotrophic cycle suggest that the adaptation to a higher lipid transport demand during oogenesis in Aedes aegypti is accomplished by increasing the amount of lipophorin in the hemolymph. This response is different from that observed in Musca domestica (Diptera) that does not involve changes in hemolymph lipophorin levels.

During Musca domestica embryogenesis, we detected a proteolytic activity that is probably involved in yolk protein degradation. This was characterized as a cathepsin B-like proteinase, with a pH optimum of 4.6 and a molecular weight of 25,000 ± 5000. An acid phosphatase activity was also found during M. domestica embryogenesis. This enzyme has a molecular weight equal to 84,300 ± 3000, is inhibited by fluoride and tartrate and therefore may be of lysosomal origin. The activity patterns of both enzymes are similar during embryogenesis. Acid phosphatase inhibition does not have any effect on the cathepsin proteolytic action against yolk protein, indicating that the two enzymes do not have a cooperative action for initiation of vitellin hydrolysis.

A larval specific high-density lipoprotein (HDL) has been isolated from Musca domestica hemolymph by a combination of density gradient and glycerol gradient ultracentrifugations. The larval lipoprotein has a density of 1.134 g/ml and is formed by at least four apoproteins with molecular weights equal to 26,000, 23,000, 21,000, and 20,000. This lipoprotein contains large amounts of hydrocarbons and phospholipids and minor amounts of diacylglycerols and cholesterol. The larval lipoprotein is completely distinct from lipophorin in regard to apoprotein composition, lipid moiety, physiological pattern, and immunological reactions. Larval lipoprotein is accumulated until the end of the feeding period. During the pupal molt this protein is utilized and is no longer detected after 2 days of pupal stadium. The results obtained imply a possible role of this protein in the puparia and/or pupal cuticle formation. Judging from the properties shown, the Musca domestica larval lipoprotein is a completely new type of insect lipoprotein.

1. Larval Musca domestica lipophorin biosynthesis was studied in vitro.2. The newly synthesized lipophorin has a density a little lower than the circulating lipophorin after 1 hr of incubation. After 3 hr of incubation the fat body cells transfer lipids to the lipophorin that attains the density of circulating lipophorin.3. The lipophorin synthesized in vitro in identical to circulating lipophorin in density and in electrophoretical behavior.4. However these two molecules must have differences since the circulating lipophorin transfers lipids to fat body cells while the synthesized in vitro does not.5. The biosynthesis of Musca lipophorin shows differences with the Manduca sexta lipophorin biosynthesis.

1. In Musca domestica haemolymph a lipid transfer particle (LTP) is present.2. Musca domestica LTP is able to catalyze the transfer of lipids between different housefly lipophorin forms and also between lipophorins of Diptera and Lepidoptera.3. The lipophorin of larval Dione juno (Lepidoptera) was purified and is composed of two apolipoproteins, apolipophorin I (M(r) = 209,000) and apolipophorin II (M(r) = 85,000) with a density of 1.124g/Ml.4. The density of housefly lipophorin undergoes variations during the gonotrophic cycle.5. The lipophorin density variation results suggest that when a high rate of lipid utilization occurs, the lipophorin has a higher density value.

Two distinct fractions of Musca domestica arylphorin were isolated by affinity chromatography on Concanavalin A-Sepharose column. The results show that in the hexameric arylphorin that do not bind to the lectin there is no Concanavalin A binding subunit and in the majority of the hexamers that bind to the lectin there is only one subunit with Concanavalin A binding site. The results indicate that the carbohydrate moiety of the arylphorin is not involved in its specific uptake by the fat bodies and integument.

1.1. Vitellogenin of Musca domestica is synthesized by fat bodies and ovaries maintained in vitro.2.2. In fat bodies, the synthesis of vitellogenin attains a maximal level at 96 hr post emergence (stage 6) while in the ovaries the maximum of vitellogenin synthesis is attained after stage 6 in the second half of the gonotrophic cycle.3.3. Since the vitellin contents of ovaries increases mainly in the second half of the gonotrophic cycle, the ovaries must be the principal source of vitellin for the eggs of M. domestica.4.4. Flies maintained on a sugar water diet show a lower, but similar, pattern of vitellogenin synthesis by the fat bodies when compared to flies maintained on a proteic diet. The main peak of vitellogenin synthesis by ovaries is dependent on administration of a proteic diet to the flies.

The storage protein of Musca domestica is taken up from the haemolymph by the fat body, during the wandering stage. Musca fat bodies incubated in vitro with radioiodinated larval haemolymph proteins incorporate storage protein. The uptake of storage protein is partially inhibited by sodium fluoride and sodium azide and also by lowering the incubation temperature.

1.1. An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum.2.2. This carotenoprotein contains: (1) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups.3.3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana.4.4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein.

The storage protein of Musca domestica is a hexameric protein with an apparent molecular weight of 500,000. The hexamers are assembled by at least three types of polypeptides: p1 and p2 with apparent molecular weights of 83,000 each, and p3 with an apparent molecular weight of 89,000.