Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes

Summary The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has M_r = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [³H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10^–6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.     

Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti

Vitelline envelope genes from the mosquito Aedes aegypti were analyzed with respect to their DNA sequences, genomic representation, temporal and spatial expression profiles and response to 20-hydroxyecdysone. Genomic clones of three vitelline envelope genes, 15a-1, 15a-2 and 15a-3 were isolated. Southern analysis indicates that all three genes are represented by a single copy in the genome. The deduced amino acid sequences of all three vitelline envelope genes contain a conserved region of 46 residues that overlaps with a region that is conserved in four Drosophila melanogaster vitelline envelope genes. DNA was sequenced flanking the 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp sequence 5′ of the 15a-2 coding region was identified with 72% identity to a sequence upstream of the Ae. aegypti VgA1 vitellogenin gene. The temporal patterns of 15a-1, 15a-2 and 15a-3 expression, as determined by Northern analysis, were similar. The spatial patterns of expression, as determined by whole-mount in situ hybridization, differed between the three genes. 15a-1 and 15a-3 were only expressed in the middle and posterior regions of the follicle, while 15a-2 was also expressed at the anterior region. Vitelline envelope gene expression was higher in ovaries that were dissected at 0, 2 and 10 h following a blood meal and then incubated in vitro for 10 h in medium containing 10⁻⁵ M 20-hydroxyecdysone, compared to ovaries that were incubated without hormone.     

The inactivation of acetylcholinesterase by alpha-terthienyl and ultraviolet light Studies in vitro and in larvae of the mosquito Aedes aegypti

Acetylcholinesterase (EC 3.1.1.7) was inactivated photochemically in solution, in the presence of dissolved terthiophene sensitizers. Alpha-terthienyl (2,2′:5,2″-terthiophene) and its isomers 3,2′:5′,2″- and 3,2′:5′,3″-terthiophenes showed very similar sensitizing properties. With all three terthiophenes, the photosensitization was completely suppressed under anaerobic conditions, and therefore the inactivation process required the presence of oxygen. The enzyme was inactivated in vivo when fourth instar larvae of the mosquito Aedes aegypti were treated with alpha-terthienyl in the presence of long-wavelength ultraviolet light. No inactivation was observed when the organisms were treated with the ultraviolet light alone, with the chemical alone, or with a previously irradiated sample of the chemical. This paper represents the first example of acetylcholinesterase inactivation in vivo by a photoactive insecticide.     

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.     

Selective transport and packaging of the major yolk protein in the sea urchin

The major yolk protein of sea urchins is an iron-binding, transferrin-like molecule that is made in the adult gut. Its final destination though is the developing oocytes that are embedded in somatic accessory cells and encompassed by two epithelial layers of the ovary. In this study, we address the dynamics of yolk transport, endocytosis, and packaging during the vitellogenic phase of oogenesis in the sea urchin by use of fluorescently labeled major yolk protein (MYP). Incorporation of MYP into the accessory cells of the ovary and its packaging into yolk platelets of developing oocytes is visualized in isolated oocytes, ovary explants, and in whole animals. When MYP is introduced into the coelom of adult females, it is first accumulated by the somatic cells of the ovarian capsule and is then transported to the oocytes and packaged into yolk platelets. This phenomenon is specific for MYP and accurately reflects the endogenous MYP packaging. We find that oocytes cultured in isolation are endocytically active and capable of selectively packaging MYP into yolk platelets. Furthermore, oocytes that packaged exogenous MYP are capable of in vitro maturation, fertilization, and early development, enabling an in vivo documentation of MYP utilization and yolk platelet dynamics. These results demonstrate that the endocytic uptake of yolk proteins in sea urchins does not require a signal from their surrounding epithelial cells and can occur autonomous of the ovary. In addition, these results demonstrate that the entire population of yolk platelets is competent to receive new yolk protein input, suggesting that they are all made simultaneously during oogenesis.     

Fundulus heteroclitus vitellogenin: The deduced primary structure of a piscine precursor to noncrystalline,liquid-phase yolk protein

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 by were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 by open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30–40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences (Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10–20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5 end and AGY codons toward the 3 end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species.Abbreviations AGY AG(T or C) - TCX TC(AGC or T) - Lv lipovitellin - Pv phosvitin - Vtg vitellogenin Correspondence to: G.J. LaFleur, Jr.     

Sequence analysis of a yolk protein secretion mutant of Drosophila melanogaster

Summary The female-sterile mutants fs(1) 1163 of Drosophila melanogaster described by Gans et al. (1975) has been characterised as a yolk protein 1 (YP1) secretion mutant (Bownes and Hames 1978b; Bownes and Hodson 1980). We have cloned and sequenced the YP1 gene from this strain, and the strain in which the mutant was induced. One amino acid substitution was found in the predicted polypeptide sequence, an isoleucine to asparagine change at position 92. The sequence of the leader peptide was identical to previously published YP1 sequences. The possible effects of the amino acid change were investigated by computer analysis, which suggests there is no major alteration of secondary structure, but that a hydrophobic region in YP1 is lost in the mutant. This may affect higher order structure.     

Cloning and expression of fat body hexamerin receptor and its identification in other hexamerin sequestering tissue of rice moth, Corcyra cephalonica

Selective receptor mediated uptake is a widely prevalent mechanism in insects by which important macromolecules are acquired. Among the various proteins sequestered by the insect fat body, the larval hexamerins form the major group. In the present work full length cDNA (2.6 kb) of hexamerin receptor with an ORF of 2.4 kb was cloned from the larval fat body of rice moth, Corcyra cephalonica. This was followed by the recombinant expression of truncated N-terminal sequence of putative hexamerin receptor and the confirmation of the expressed recombinant protein as the truncated hexamerin receptor by ligand blot analysis. Apart from this we also analyzed other hexamerin sequestering tissues like salivary gland, male accessory reproductive gland and ovary for the presence of hexamerin receptor. We found that the receptor in these tissues was similar in size and mode of activation to that of fat body hexamerin receptor, thus cementing the fact that identical hexamerin receptors are present in all the hexamerin sequestering tissues in the rice moth.     

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.     

Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna

Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.