Vg Receptor Research Articles

Triazophos-induced vertical transmission of rice stripe virus is associated with host vitellogenin in the small brown planthopper Laodelphax striatellus.

Rice stripe virus (RSV) is one of the most serious rice diseases in East Asia. The small brown planthopper (SBPH), Laodelphax striatellus, is an economically important rice pest. SBPH transmits RSV horizontally and vertically, resulting in serious rice economic losses. Exposure to sublethal doses of insecticides can induce many physiological and cellular changes in insects. However, the mechanism underlying triazophos-induced RSV vertical transmission remains unclear. Here, we investigated the role of vitellogenin (Vg) in triazophos-induced RSV vertical transmission. RSV vertical transmission rates were significantly induced by sublethal exposure to triazophos. The transcript abundance of viral RNA3 segment (RNA3) and capsid protein (CP) in the ovaries of SBPH were also significantly increased. Triazophos induced the expression level of Vg in L. striatellus (LsVg) and increased the contents of ovarian protein and fat body protein. Knockdown of Vg significantly reduced the expression levels of LsVg and Vg receptor (LsVgR), and decreased RSV accumulations in the ovaries. Double-stranded Vg (dsVg)-mediated down-regulation could be rescued by exposure to triazophos. Vertical transmission rate of the dsVg-injected group was significantly decreased compared with the dsGFP-injected group, and triazophos significantly rescued the RSV vertical transmission rate of the dsVg-injected group. Our results indicate that triazophos-induced RSV vertical transmission is associated with Vg. This work will help us to further elucidate sublethal doses of insecticides-mediated effects and develop new strategies for pest control. © 2019 Society of Chemical Industry.

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis.

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.

Alteration of insulin and nutrition signal gene expression or depletion of Met reduce both lifespan and reproduction in the German cockroach

In insects, nutrition and hormones coordinately regulate lifespan and reproduction, which might affect each other. We here investigated how nutrition, insulin, and juvenile hormone (JH) signal genes affect lifespan and reproduction in the German cockroach, Blattella germanica, a serious urban pest throughout the world. Starvation as well as altering insulin and nutrition signal genes by RNA interference (RNAi) knockdown of the expression of either positive or negative components in the two pathways simultaneously reduced lifespan and ootheca number of the mated female cockroaches. Meanwhile, depletion of the JH receptor Methoprene-tolerant (Met), but not kruppel homolog 1 (Kr-h1) that predominately transduces JH signaling to prevent metamorphosis, significantly reduced the two parameters. Moreover, suppressing the expression of several reproduction-related genes, including doublesex (Dsx), vitellogenin (Vg), and the Vg receptor (VgR), had little yet various effects on lifespan; nevertheless, it is likely that there are some reproduction-independent mating factors reducing lifespan. In conclusion, through blocking lifespan and reproduction in a simultaneous manner, the alteration of insulin and nutrient signal gene expression or the depletion of Met might provide a great potential for controlling the German cockroach.

Insulin signaling mediates previtellogenic development and enhances juvenile hormone-mediated vitellogenesis in a lepidopteran insect, Maruca vitrata

BackgroundInsulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females.ResultsA pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis.ConclusionsIIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Intracellular localization of vitellogenin receptor mRNA and protein during oogenesis of a parthenogenetic tick, Haemaphysalis longicornis

BackgroundVitellogenin (Vg), a key molecule for oocyte development synthesized in the fat body during blood-feeding, is released into the hemolymph and then taken into the oocytes via Vg receptor (VgR) in ticks. Previously, we showed that VgR mRNA is expressed in the ovary at the adult stage of parthenogenetic Haemaphysalis longicornis ticks and its expression increases after blood-feeding. However, intracellular localization of VgR mRNA and protein at each developmental stage of oocytes during oogenesis remains largely unclear.MethodsmRNA and protein expression profiles of H. longicornis VgR (HlVgR) in the oocytes from the unfed to oviposition periods were analyzed by real-time PCR, in situ hybridization, and immunostaining. To elucidate the timing of the onset of Vg uptake, RNA interference (RNAi)-mediated gene silencing of HlVgR was performed.ResultsIn situ hybridization revealed that HlVgR mRNA was detected in the cytoplasm of stage I-III oocytes, and weaker positive signals for HlVgR mRNA were found in the cell periphery of stage IV and V oocytes. Likewise, HlVgR protein was detected by immunostaining in the cytoplasm of stage I-III oocytes and in the cell periphery of stage IV and V oocytes. Each developmental stage of the oocytes showed distinct patterns of mRNA and protein expression of HlVgR. Moreover, RNAi of HlVgR caused delayed or arrested development in the oocytes. The ovaries of control ticks showed all developmental stages of oocytes, whereas stage I-III oocytes were found in the ovaries of HlVgR-RNAi ticks at 5 days after engorgement.ConclusionsThese results suggest that active uptake of Vg is required for development from stage III to stage IV during oogenesis. Our data clearly revealed an apparent shift in the intracellular localization of VgR for both mRNA and protein level in oocytes during oogenesis.

Rice stripe virus hitchhikes the vector insect vitellogenin ligand-receptor pathway for ovary entry.

It is known that plant arboviruses infect insect vector cells by endocytosis; however, the cellular receptors that mediate endocytosis have not been well defined. In our recently published work and this study, by clarifying the vertical transmission mechanism of Rice stripe virus (RSV) in Laodelphax striatellus, we provide a novel paradigm for how arboviruses enter insect germ-line cells. Instead of direct interaction with a viral receptor, the virus binds to a secreted ligand protein, hitchhiking the ligand-receptor pathway to achieve cell entry. Vitellogenin (Vg) is an indispensable protein for embryo development that is synthesized extra-ovarially and taken up by germ-line cells through Vg receptor (VgR)-mediated endocytosis. After revealing that RSV invades L. striatellus ovary by a specific molecular interaction with the insect Vg in haemolymph, this study addressed VgR's function in mediating the RSV invasion of the germarium nurse cells, further confirming the ligand's receptor-mediated viral cell-invasion mechanism. Understanding the viral ovary-entry pathways in vectors will help to find suitable measures to block the trans-generation transmission of the viruses. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

The cholesterol-hydroxyecdysone-vitellogenin pathway is involved in the longevity of trophocytes and oenocytes of queen honey bees (Apis mellifera)

Trophocytes and oenocytes in the abdomen of honey bees do not divide after eclosion; however, trophocytes and oenocytes of queen bees have a longer lifespan and maintain better cellular function than those of worker bees. To explore this phenomenon, we assayed the molecules involved in the cholesterol-hydroxyecdysone-vitellogenin (Vg) pathway in the trophocytes and oenocytes of young and old worker and queen bees. The results showed that Vg and cholesterol levels in hemolymph and cholesterol levels, 20-hydroxyecdysone (20E) levels, and the messenger RNA levels of cytochrome P450 314A1 20-hydroxylase (Cyp314A1), ecdysone receptor isoform A (EcR-A), ecdysone receptor isoform B1 (EcR-B1), ultraspiracle (USP), ecdysone-induced protein 74 (E74), ecdysone-induced protein 75 (E75), broad-complex (BR-C), Vg, and Vg receptor (VgR) in trophocytes and oenocytes were increased in queen bees compared with worker bees. These findings indicated that queen bees have higher expression of molecules in the cholesterol-hydroxyecdysone-Vg pathway than worker bees.

A NONOVARY-SPECIFIC VITELLOGENIN RECEPTOR FROM THE ORIENTAL FRUIT FLY, Bactrocera dorsalis (HENDEL).

The yolk protein precursor, vitellogenin (Vg), is absorbed into growing oocytes via receptor-mediated endocytosis for embryonic development. In this study, a Vg receptor (VgR) cDNA of the oriental fruit fly (Bactrocera dorsalis Hendel) was cloned via RT-PCR and RACE (GenBank accession no. KR535603) and its expression analyzed. The BdVgR cDNA has a length of 6,585 bp encoding 1,923 amino acids. It has a conserved motif arrangement with other insect VgRs, and showed high identity to the B. cucurbitae VgR (91.4%). The expression of BdVgR mRNA and proteins was shown in both ovary and fat body. This is the first report on a nonovary-specific VgR from a nonsocial insect. In ovary, the expression of BdVgR mRNA and proteins was inconsistent, with the transcription, but not protein, level high on D0. In fat body, the expression levels of BdVgR mRNA and proteins were high on days 5 and 6. The function of BdVgR in the fat body is not clear. However, it may be involved in reuptake of yolk proteins from the hemolymph as an amino acid reservoir or as autocrine regulation of yolk protein expression.

Four cDNAs encoding lipoprotein receptors from shrimp (Pandalopsis japonica): Structural characterization and expression analysis during maturation

As in all other oviparous animals, lipoprotein receptors play a critical role in lipid metabolism and reproduction in decapod crustaceans. Four full-length cDNAs encoding lipoprotein receptors (Paj-VgR, Paj-LpR1, Paj-LpR2A, and Paj-LpR2B) were identified from Pandalopsis japonica through a combination of EST screening and PCR-based cloning. Paj-LpR1 appears to be the first crustacean ortholog of insect lipophorin receptors, and its two paralogs, Paj-LpR2A and Paj-LpR2B, exhibited similar structural characteristics. Several transcriptional isoforms were also identified for all three Paj-LpRs. Each expression pattern was unique, suggesting different physiological roles for these proteins. Paj-VgR is an ortholog of vitellogenin (Vg) receptors from other decapod crustaceans. A phylogenetic analysis of lipoproteins and their receptors suggested that the nomenclature of Vgs from decapod crustaceans may need to be changed. A PCR-based transcriptional analysis showed that Paj-VgR and Paj-LpR2B are expressed almost exclusively in the ovary, whereas Paj-LpR1 and Paj-LpR2A are expressed in multiple tissues. The various transcriptional isoforms of the three Paj-LpRs exhibited unique tissue distribution profiles. A transcriptional analysis of each receptor using tissues with different GSI values showed that the change in transcription of Paj-VgRs, Paj-LpR2A and Paj-LpR1 was not as significant as that of Vgs during maturation. However, the transcriptional levels of Paj-LpR2B decreased in ovary at maturation, suggesting that their transcriptional regulation is involved in reproduction.

Trans-order yolk protein can stimulate endocytic activity in Drosophila oocytes

Vitellogenins (Vgs) and mature yolk from some non-Dipteran insects can be recognized by Drosophila melanogaster oocyte Vg receptors and incorporated via receptor-mediated endocytosis into nascent yolk spheres (NYS). It had previously been assumed that only Vgs of Drosophila or other Dipterans could be so endocytosed. Drosophila ovarian follicles from 4-day old females were incubated in the presence of physiological salt solution (PSS) containing some fluorescent TexasRed-Dextran (Dex-red) or PSS-Dex-red in which either female hemolymph, or vitellin (mature yolk) from lysed oocytes was present from any of the following: (1) Drosophila (Diptera); (2) Oncopeltus (milkweed bug, Hemiptera); (3) Acteaus (luna moth, Saturniidae Lepidoptera); (4) Papilio (swallowtail butterfly, Papilionidae Lepidoptera); or (5) Xylocopa (carpenter bee, Hymenoptera). Under incubation conditions, any NYS would become fluorescent due to non-specific fluid-phase uptake. Ovarian follicles incubated in PSS-DexRed alone or in PSS with hemolymph from males did not carry out endocytosis detectable by this technique, but all other treatments listed above did.

Cloning and characterization of the autophagy-related gene 6 from the hard tick, Haemaphysalis longicornis

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.

From Hepatopancreas to Ovary: Molecular Characterization of a Shrimp Vitellogenin Receptor Involved in the Processing of Vitellogenin1

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3-4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.

Hormonal regulation of metamorphosis and reproduction in ticks

The presence of a "status quo" hormone like JH has not been found in ticks. The most advanced understanding of tick endocrinology is associated with female reproduction, where the sequence of the first messages for storage proteins (vitellogenin (Vg) and carrier protein), the Vg receptor, and male peptidic pheromones were recently reported. The current consensus model suggests that ecdysteroids from the epidermis regulated by a putative peptidic ecdysiotrophic hormone from the synganlion initiates the expression of the Vg messages in fat body and midgut. Vg protein, secreted into the hemolymph, requires an ovary Vg receptor to be absorbed by oocytes. Male pheromones transferred into the female genital tract during mating initiate blood feeding to repletion and vitellogenesis. The work so far on tick endocrinology is limited by the paucity of identified hormones and the small number of studies on a few tick models. The role of storage proteins in the evolution of hematophagy is discussed.

Instability of crab vitellogenin and its immunological relatedness with mammalian atherogenic lipoproteins.

Vitellogenesis is the process of accumulation of vitellogenin (Vg) in rapidly growing oocytes of oviparous animals and its' subsequent transformation into lipovitellin (Lv). Lipovitellin, which forms the major yolk protein, serves as a principal nutrient reserve for the developing embryo. In the present study, Vg and Lv were purified from the hemolymph and ovary, respectively of the crab Scylla serrata by gel filtration followed by preparative gel electrophoresis. It was observed that purified Vg, but not Lv, possessed an intrinsic protease activity with which it underwent autoproteolysis giving rise to several smaller proteins. Furthermore, urea-mediated unfolding studies by UV-spectral analysis revealed clearly that Vg was easily disrupted by urea whereas Lv was resistant. Taken together, these results suggest that although Lv had a stable conformation, its precursor Vg was labile and highly sensitive to degradation. Another aspect that was investigated in the present study was the immunological kinship of crab Vg and Lv to mammalian atherogenic lipoproteins, the low density lipoprotein (LDL), very low density lipoprotein (VLDL), and apolipoprotein B (apoB). By Western blot analysis, it was demonstrated that crab Vg and Lv were immunoreactive to antibodies to human LDL, VLDL, and apoB. These observations suggest the existence of common epitope recognition sites in crab Vg and mammalian lipid transferring proteins. This corroborates well with our earlier study on the recognition of crab Vg receptor by mammalian lipoproteins.

Oocyte growth in temperate basses: multiple forms of vitellogenin and their receptor

A full-length cDNA encoding a vitellogenin (Vg) receptor (VgR) was isolated and sequenced from a white perch, Morone americana, ovarian cDNA library. The perch expresses a single VgR that is structurally classified as an LR8 type (without an 0-linked sugar domain) and appears to be the only species observed to possess no splice variant of the receptor. We proposed a hypothetical model for ovarian lipid transfer in Morone species, one in which the `single VgR' transports Vg and other lipoproteins via a selective endocytotic pathway based on ligand affinity, in conjunction with a lipase-mediated pathway of fatty acid transport.

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins.

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.

Ovarian follicle growth, maturation, and ovulation in teleost fish

Knowledge of ovarian follicle growth, maturation, and ovulation in teleosts is important to many aspects of basic and biomedical research, fisheries management, aquaculture, and environmental science. Follicle growth includes previtellogenic and vitellogenic stages. Although little progress has been made in our understanding of previtellogenic development, information on the regulation and mechanisms of vitellogenic growth has increased substantially in recent years. This new information pertains to the multiplicity of vitellogenin (Vg) genes, the oocyte Vg receptor, Vg processing enzymes (cathepsins) and their specific yolk products, and synthesis of various vitelline envelope precursor proteins in liver and ovary. Oocyte acquisition of lipids from sources other than Vg dominates follicle growth in many teleosts, especially perciformes, yet our understanding of the basic processes involved remains rudimentary. Considerable advances also have been made in our understanding of the endocrine regulation and mechanisms of ovarian follicle maturation. We have learned that ovarian follicle maturation involves a number of events including luteinizing hormone (LH)-dependent acquisition of oocyte maturational competence, LH induction of maturation-inducing hormone (MIH) synthesis, and MIH-dependent meiotic resumption (nuclear maturation) and cytoplasmic maturation. While much has been learned about mechanisms of maturational competence, MIH synthesis and meiotic resumption, our knowledge of cytoplasmic maturation is limited to descriptions of MIH-dependent yolk protein hydrolysis and associated oocyte hydration in a few species. It has become apparent that ovulation requires genomic activation regulated by nuclear MIH receptors, but the transduction pathways for the ovulatory MIH signal appear to be complex and are poorly understood.

Molecular characteristics of insect vitellogenins and vitellogenin receptors

The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent β-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.

Characterization of the solubilized mosquito vitellogenin receptor

In this study we solubilized and characterized the receptor for the major egg yolk protein precursor, vitellogenin (Vg), from the yellow fever mosquito, Aedes aegypti. The receptor was solubilized from vitellogenic ovary membranes using octyl-β- d-glucoside (OG). Under equilibrium binding conditions, [ 35S]Vg bound with high affinity ( K d = 2.8 × 10 −8 M) to a single class of binding sites in solubilized ovary extracts. The solubilized receptor was present in ovarian extracts and bound selectively A. aegypti Vg and its storage form, vitellin (Vn). The receptor preparation was heat and trypsin sensitive. Binding of Vg to its receptor could be inhibited as well dissociated with suramin. The receptor was visualized by ligand-blotting as a 205 kDa protein under non-reducing conditions. It did not share immunological cross-reactivity with antibodies to chicken and locust Vg receptors. Vitellogenin, Vn and its purified subunits competed for binding to the receptor in the order, Vg ≈ Vn > Vn large subunit > Vn small subunit. Binding of dephosphorylated Vg was significantly reduced. Deglycosylated Vg, on the other hand, formed high molecular weight aggregates resulting in artifactually high binding which indicates importance of glycosylation for the stability of Vg molecule. During egg maturation, the number of receptor binding sites in ovaries correlated with the rate of Vg uptake and peaked between 24–30 h after which it reduced to no binding by 48 h post blood meal.

Binding of vitellogenin to membranes isolated from mosquito ovaries

The presence of specific receptors for vitellogenin (Vg) in ovary membranes of the mosquito, Aedes aegypti, was demonstrated by an in vitro binding assay. The binding reaction, which is dependent on pH and Ca2+, uses 4 micrograms membrane protein, 35S-Vg labeled metabolically by fat body culture in vitro, and unlabeled vitellin (Vn) for competition. At pH 7.0 and in the presence of 5 mM Ca2+, the binding of Vg to its receptor reaches equilibrium within 60-90 min at both 4 and 25 degrees C. The binding is specific to membranes prepared only from ovaries. While mosquito Vg and Vn bind with equal affinity to Vg receptors on ovary membranes, neither locust Vg nor mouse IgG has any measurable affinity towards these sites. Nonlinear least square analysis of the saturation isotherms is consistent with the presence of a single class of Vg receptors on ovary membranes with a dissociation constant (Kd) of 0.18 microM.