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.     

Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse-labeling of lipophorin with [³⁵S]methionine showed that apolipophorin-I and -II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse-labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m-chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin-I or -II. Incubation of fat bodies with [³H]oleate resulted in the secretion of lipophorin containing [³H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [³H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [³H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin-associated lipids and secretion of lipids mediated by the lipid-transfer particle outside the plasma membrane of the fat body.     

Isolation and sequence of a partial vitellogenin cDNA from the cockroach,Blattella germanica (L.) (Dictyoptera,Blattellidae), and characterization of the vitellogenin gene expression

A partial cDNA clone of the vitellogenin gene from the cockroach Blattella germanica has been isolated from a cDNA expression library using an anti-vitellin–vitellogenin antiserum probe. The analysis of cDNA inserts gave a sequence of 2,645 nucleotides corresponding to the 3′ region. The deduced amino acid sequence is 825 residues long and is similar to the homologous portion of the vitellogenin of other insect species, especially that of the mosquito Aedes aegypti. RNA hybridization studies indicated that the vitellogenin gene expression is limited to the fat body of adult females. The pattern of expression during the first vitellogenic cycle was approximately parallel to that of vitellogenin production by the fat body previously described. The availability of a cDNA probe for the B. germanica vitellogenin gene represents a useful tool to study the molecular action of hormones affecting vitellogenin synthesis in this species. Arch. Insect Biochem. Physiol. 38:137–146, 1998. © 1998 Wiley-Liss, Inc.     

Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

Injected β-ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 μg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0·5 μg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.     

Role of the fat body in the regulation of host-seeking behaviour in the mosquito,Aedes aegypti

Following a blood meal that initiates oöcyte development, the host-seeking behaviour of Aedes aegypti mosquitoes is inhibited by a haemolymph-borne factor that is released in response to a humoral signal from a vitellogenic ovary. This inhibition is accompanied by a decrease in the sensitivity of the peripheral lactic acid receptors. Implantation of corpora allata, medial neurosecretory cells, or terminal abdominal ganglia from blood-fed donors could not induce the inhibition in sugar-fed recipients. However, fat body transplanted from blood-fed into sugar-fed females suppressed host-seeking behaviour as well as the sensitivity of lactic acid receptors, suggesting that the source of the behavioural inhibitor is the fat body. Resting-stage ovaries from other mosquito species inhibited host-seeking after the A. aegypti host was fed on blood only if the fat body was activated by the donor ovary.     

Lipophorin levels in the yellow fever mosquito,Aedes aegypti,and the effect of feeding

High density lipophorin (HDLp) is the major lipid transport vehicle in insect hemolymph. Using an indirect ELISA, levels of HDLp were measured in the yellow fever mosquito, Aedes aegypti. The level of lipophorin, when normalized to the total weight of the insect, was similar in the different developmental stages. Starvation (access to water only) of adult females did not affect the level of HDLp nor its density when compared to sugar-fed females. On the other hand, blood feeding (of normally sugar-fed females) resulted in a three-fold increase of the HDLp level at 40 h after feeding. This increase was accompanied by a slight but significant increase in the density of HDLp at 24 h after feeding. Ingestion of a lipid-free protein meal or a lipid-supplemented protein meal induced changes in HDLp level and density that were comparable to those induced by ingestion of a blood meal. Ingestion of a blood meal, following starvation (access to water only) from the moment of adult emergence, did not induce an increase in HDLp level. The results presented indicate that, in contrast to other insect species, A. aegypti responds to an increased need for lipid transport in the hemolymph by increasing the amount of HDLp. Arch. Insect Biochem. Physiol. 34:301–312, 1997. © 1997 Wiley-Liss, Inc.     

A novel function of 20-hydroxyecdysone: translational repression of the lysosomal protease mRNA in the mosquito fat body

In the female fat body of the mosquito Aedes aegypti, lysosomes play important roles during the cessation of vitellogenesis by degrading the biosynthetic machinery and aiding the remodeling of the fat body cells. A detailed study of a mosquito lysosomal aspartic protease (AaLAP) has shown a unique expression pattern in the vitellogenic fat body: the level of AaLAP mRNA dramatically rises and peaks at 24 h post blood meal (PBM) correlating with the high titer of ecdysteroids; however, there is a 12 h lag before peak levels of AaLAP protein and its enzymatic activity has been observed. These observations suggest that the high titer of 20-hydroxyecdysone (20E) may hinder translation of the AaLAP mRNA. Here, we used an in vitro organ culture to study the effect of 20E on the protein synthesis of AaLAP in the fat body. The increase in the AaLAP protein level in the fat body, dissected at 24 h PBM and incubated for 6 or 12 h, was inhibited by the presence of 10(-5) M 20E in the medium. Incubation in the hormone-free medium did not effect accumulation of the AaLAP protein which proceeded at the levels comparable to the intact insect. Furthermore, the effect of 10(-5) M 20E on the AaLAP accumulation was reversible. These experiments support the hypothesis of the 20E-mediated repression of lysosomal protease mRNAs at the translational level in the regulation of vitellogenic and postvitellogenic events in the mosquito fat body. Analysis of the 5' and 3' -end untranslated regions (UTR) of AaLAP mRNA form secondary structures suggest that they may also contribute to mRNA stability and 20E-mediated translational inhibition.     

Ploidy levels and DNA synthesis in fat body cells of the adult mosquito,Aedes aegypti: The role of juvenile hormone

Adult females of the mosquito Aedes aegypti showed two cycles of DNA replication in the fat body based on microspectrophotometric measurement of changes in nuclear DNA. The first cycle began after emergence and resulted in 80% of diploid fat body cells becoming tetraploid and 20% becoming octoploid by the end of the third day. The second replication cycle occurred 48–72 h after a blood meal and resulted in an increase in octoploid nuclei to 67% Topical application of juvenile hormone or methoprene to abdomens isolated at emergence stimulated an increase in ploidy levels above that normally seen in situ. Synthesis of DNA, estimated by incorporation of injected [³H]-thymidine, rose after emergence and remained high for 2 days. Synthesis increased again after a blood meal, reached a peak by 6 h, and returned to low levels by 24 h after the meal. The timing of DNA synthesis and a measurable increase in ploidy were temporally separated. The ploidy increase, but not DNA synthesis, was correlated with increases in juvenile hormone levels.     

A Critical Role of the Nuclear Receptor HR3 in Regulation of Gonadotrophic Cycles of the Mosquito Aedes aegypti

The orphan nuclear receptor HR3 is essential for developmental switches during insect development and metamorphosis regulated by 20-hydroxyecdysone (20E). Reproduction of female mosquitoes of the major vector of Dengue fever, Aedes aegypti, is cyclic because of its dependence on blood feeding. 20E is an important hormone regulating vitellogenic events in this mosquito; however, any role for HR3 in 20E-driven reproductive events has not been known. Using RNA interference (RNAi) approach, we demonstrated that Aedes HR3 plays a critical role in a timely termination of expression of the vitellogenin (Vg) gene encoding the major yolk protein precursor. It is also important for downregulation of the Target-of-Rapamycin pathway and activation of programmed autophagy in the Aedes fat body at the end of vitellogenesis. HR3 is critical in activating betaFTZ-F1, EcRB and USPA, the expressions of which are highly elevated at the end of vitellogenesis. RNAi depletion of HR3 (iHR3) prior to the first gonadotrophic cycle affects a normal progression of the second gonadotrophic cycle. Most of ovaries 24 h post second blood meal from iHR3 females in the second cycle were small with follicles that were only slightly different in length from of those of resting stage. In addition, these iHR3 females laid a significantly reduced number of eggs per mosquito as compared to those of iMal and the wild type. Our results indicate an important role of HR3 in regulation of 20E-regulated developmental switches during reproductive cycles of A. aegypti females.     

Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Lp(a) glycoprotein exhibits an apparent size polymorphism that is associated with genetically controlled Lp(a) lipoprotein concentrations in plasma (Utermann et al. 1988). We have tested the hypothesis that this polymorphism is genetically controlled by studying 15 matings with a total of 44 offspring. This confirmed our conclusion that Lp(a) types are controlled by a series of codominant alleles Lp^F, Lp^B, Lp^S1, Lp^S2, Lp^S3 and Lp^S4 and by a null allele Lp^o. Together with the data from the accompanying paper this indicates that the structural gene for the Lp(a) protein is the major gene locus determining Lp(a) lipoprotein concentrations in plasma.     

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.     

In vitro synthesis and secretion of lipophorin by the fat body of nondiapause and prediapause larvae of the southwestern corn borer,Diatraea grandiosella

The capacity of the fat body of nondiapause, prediapause and diapause larvae of the southwestern corn borer, Diatraea gradiosella, to synthesize and release lipophorin was examined in vitro using [³H]leucine as the radiotracer. Synthesis and release of [³H]lipophorin by the fat body peaked in 11–13 day-old fifth instar nondiapause larvae, which coincided with their feeding period. The rate of lipophorin synthesis in the fat body of newly ecdysed pupae was extremely low. Synthesis and release of [³H]lipophorin by the fat body of prediapause larvae occurred at the highest rates in 20–35 day-old fifth and sixth instars, and declined to virtually undetectable levels after larvae entered diapause around 40 days-of-age. Immunoprecipitation of [³H]lipophorin from fat body of 13 day-old nondiapause larvae that had been pulse-labeled with [³H]leucine showed that the half life of lipophorin synthesis and processing was about 40 minutes. Release of total protein and lipophorin from the fat body of 13 day-old nondiapause larvae into Grace's medium was inhibited by 56 and 60%, respectively, when 10 μg/ml tunicamycin was incorporated into medium.