Insect Vitellogenin Research Articles

Testosterone maintains male longevity and female reproduction in Chrysopa pallens

Vertebrate testosterone, an androgen present in the testes, is essential for male fertility. Vertebrate-type steroid hormones have been identified in insects, but their function remains unknown. Insect vitellogenin (Vg) is usually a female-specific protein involved in reproductive processes. However, males of some species, such as the green lacewing Chrysopa pallens, have Vg. Here, we demonstrated that the knockdown of C. pallens male Vg by RNAi significantly shortened the lifespan of males, suppressed the reproduction of post-mating females, and strikingly reduced the abundance of several immune-related compounds, including testosterone. LC-MS/MS revealed that C. pallens male testosterone had the same structure and molecular mass as vertebrate testosterone. Topical testosterone application partially restored the lifespan of Vg-deficient males and the reproduction of post-mating females. These results suggest that vertebrate-type testosterone maintains male longevity and female reproduction under the control of the male Vg in C. pallens.

Unusual functions of insect vitellogenins: minireview.

Insect vitellogenins are an intriguing class of complex proteins. They primarily serve as a source of energy for the developing embryo in insect eggs. Vitellogenesis is a complex hormonally and neurally controlled process that command synthesis of vitellogenin molecules and ensures their transport from the female fat bodies or ovarial cells into eggs. The representatives of all insect hormones such as juvenile hormones, ecdysteroids, and neurohormones participate in vitellogenesis, but juvenile hormones (most insect species) and ecdysteroids (mostly Diptera) play the most important roles in the process. Strikingly, not only insect females, but also males have been reported to synthesize vitellogenins indicating their further utility in the insect body. Indeed, it has recently been found that vitellogenins perform a variety of biological functions in the insect body. They participate in defense reactions against entomopathogens such as nematodes, fungi, and bacteria, as well as against venoms such as the honeybee Apis mellifera venom. Interestingly, vitellogenins are also present in the venom of the honeybee itself, albeit their exact role is unknown; they most likely increase the efficacy of the venom in the victim's body. Within the bee's body vitellogenins contribute to the lifespan regulation as anti-aging factor acting under tight social interactions and hormonal control. The current minireview covers all of these functions of vitellogenins and portrays them as biologically active substances that play a variety of significant roles in both insect females and males, and not only acting as passive energy sources for developing embryo.

Elevation of vitellogenin expression in Sunn pest is associated with pre‐migration and migration phases of the life cycle

AbstractSunn pest, Eurygaster maura L. (Hemiptera: Scutelleridae), a species with an obligatory diapause, is a major destructive pest of cereal products in central Asia, Europe and North Africa. Adults feed voraciously, causing total destruction of wheat fields in just a couple of days. Insect vitellogenins (Vgs) play a major role in reproduction by supplying the resources needed for oocyte development. In this study, we identified and characterized three E. maura Vg genes (EmVgs: EmVg1, EmVg2 and EmVg3) in the cDNA library generated from the fat body of overwintering E. maura. We examined expression levels of EmVgs in the phases of the life cycle, different tissues and at different developmental stages. mRNA levels in the female adults started to increase in the pre‐migration phase and reached peak level in the adults that freshly migrated to lowlands. This pattern suggests that the elevation of EmVg expression is associated with pre‐migration and migration phases of the insect's life cycle. EmVgs were primarily expressed in the fat body, which could be a possible EmVg expression site. EmVgs were expressed throughout developmental stages, suggesting that they might be prominent for nymphal development of E. maura.

Molecular cloning, expression profiling and RNA interference of a vitellogenin gene from Harmonia axyridis (Coleoptera: Coccinellidae)

In this study, the Harmonia axyridis vitellogenin gene 2 (HmVg2) sequence was identified from transcriptomic databases of female adult H. axyridis and cloned into pMD18-T vector. The HmVg2 gene was 5460 bp in length, and formed with an open reading frame (ORF) of 5 361 nucleotides (GenBank accession no. KY794939). The putative molecular weight of the primary HmVg2 protein was 203.459 kDa and the predicted isoelectric point (pI) was 8.59. HmVg2 contained a signal peptide, vitellogenin N-terminal (vitellogenin-N) domain, domain of unknown function 1943 (DUF1934) domain and von Willebrand factor type D (VWD) domain. The developmental expression profiling showed that HmVg2 was extremely highly expressed in female insects, but was expressed at lower levels in male insects. In female insects, HmVg2 was mainly expressed in the wing and fat body. The double-stranded RNA-HmVg2/-GFP was injected into H. axyridis, and qRT-PCR results showed that the HmVg2 gene was specifically silenced. The eggs laid during the first five days and the hatching rate of eggs was lower than controls after dsHmVg2 injection. This investigation demonstrated that the HmVg2 gene plays an important role in H. axyridis reproduction and enriches the function of the insect vitellogenin gene.

Molecular cloning, structural analysis, and the developmental expression of vitellogenin in the giant mealworm Zophobas atratus

Abstract The giant mealworm Zophobas atratus, which is used for commercial purposes, was officially introduced to Korea. However, physiological information of oocyte maturation for the mass rearing of Z. atratus has not been investigated. Here, we cloned Z. atratus vitellogenin (ZaVg) cDNA and analyzed the expression profiles during development to better understand oocyte maturation in Z. atratus. A multi-alignment and phylogenetic analysis using the deduced amino acids of ZaVg and those of other insect vitellogenins showed that ZaVg has typical Coleopteran Vg features. Consistent with ovarian development, lower expression levels of ZaVg protein in the fat body, hemolymph, and ovary were observed at 0 and 10 days in the adult stage, while higher expression levels of that were detected at 20, 30, and 40 days in the adult stage. These results suggest that the expression level of ZaVg is adult-stage specific, which providing basic information on Vg and oocyte development in Z. atratus.

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'.

Endocrinologia e controle da vitelogênese em carrapatos

Background : : : : Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review : It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion : Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.

Insect tissue-specific vitellogenin facilitates transmission of plant virus.

Insect vitellogenin (Vg) has been considered to be synthesized in the fat body. Here, we found that abundant Vg protein is synthesized in Laodelphax striatellus hemocytes as well. We also determined that only the hemocyte-produced Vg binds to Rice stripe virus (RSV) in vivo. Examination of the subunit composition of L. striatellus Vg (LsVg) revealed that LsVg was processed differently after its expression in different tissues. The LsVg subunit able to bind to RSV exist stably only in hemocytes, while fat body-produced LsVg lacks the RSV-interacting subunit. Nymph and male L. striatellus individuals also synthesize Vg but only in hemocytes, and the proteins co-localize with RSV. We observed that knockdown of LsVg transcripts by RNA interference decreased the RSV titer in the hemolymph, and thus interfered with systemic virus infection. Our results reveal the sex-independent expression and tissue-specific processing of LsVg and also unprecedentedly connect the function of this protein in mediating virus transmission to its particular molecular forms existing in tissues previously known as non-Vg producing.

Ancient Duplications Have Led to Functional Divergence of Vitellogenin-Like Genes Potentially Involved in Inflammation and Oxidative Stress in Honey Bees.

Protection against inflammation and oxidative stress is key in slowing down aging processes. The honey bee (Apis mellifera) shows flexible aging patterns linked to the social role of individual bees. One molecular factor associated with honey bee aging regulation is vitellogenin, a lipoglycophosphoprotein with anti-inflammatory and antioxidant properties. Recently, we identified three genes in Hymenopteran genomes arisen from ancient insect vitellogenin duplications, named vg-like-A, -B, and -C. The function of these vitellogenin homologs is unclear. We hypothesize that some of them might share gene- and protein-level similarities and a longevity-supporting role with vitellogenin. Here, we show how the structure and modifications of the vg-like genes and proteins have diverged from vitellogenin. Furthermore, all three vg-like genes show signs of positive selection, but the spatial location of the selected protein sites differ from those found in vitellogenin. We show that all these genes are expressed in both long-lived winter worker bees and in summer nurse bees with intermediate life expectancy, yet only vg-like-A shows elevated expression in winter bees as found in vitellogenin. Finally, we show that vg-like-A responds more strongly than vitellogenin to inflammatory and oxidative conditions in summer nurse bees, and that also vg-like-B responds to oxidative stress. We associate vg-like-A and, to lesser extent, vg-like-B to the antiaging roles of vitellogenin, but that vg-like-C probably is involved in some other function. Our analysis indicates that an ancient duplication event facilitated the adaptive and functional divergence of vitellogenin and its paralogs in the honey bee.

Osmia cornifrons vitellogenin: cDNA cloning, structural analysis and developmental expression

AbstractOsmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. Osmia cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. Osmia cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C‐terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus and 42%–30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachilidae, Apidae, Vespidae and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species. The expression profile of O. cornifrons vitellogenin mRNA during development revealed that O. cornifrons vitellogenin was first detected in the pupal stage and was continuously detected during the adult stage. Interestingly, O. cornifrons vitellogenin mRNA expression was low in mid‐diapause, then gradually increased beginning on day 3 of the newly emerged adult stage, and subsequently declined. These results suggest that the expression level of O. cornifrons vitellogenin mRNA is stage‐specific.

Evolutionary conservation of amino acid composition in paralogous insect vitellogenins

Comparison of paralogous vitellogenins in 10 insect species representing six orders showed a remarkable degree of conservation of amino acid composition in spite of sequence differences. For example, the correlation between the percentages of the 20 amino acids in two vitellogenins from the beetle Tribolium castaneum was 0.975, even though the two amino acid sequences differed from each other at 49.4% of sites. There was a positive correlation between the frequency of occurrence of reciprocal pairs of amino acids in more distantly related paralogs, and this correlation was generally strongest when both of the amino acids in the pair were nutritionally essential. These results imply that conservation of amino acid composition occurs through amino acid replacements that result in a balanced loss and gain of each amino acid residue. Thus insect vitellogenins seem to be subject to an unusual kind of purifying selection, where the amino acid content is conserved rather than the sequence per se, selection apparently arising from the nutritional needs of the developing embryo appears to be responsible for maintaining the balance of amino acids.

Tissue- and Stage-specific Expression of Two Lipophorin Receptor Variants with Seven and Eight Ligand-binding Repeats in the Adult Mosquito

We identified two splice variants of lipophorin receptor (LpR) gene products specific to the mosquito fat body (AaLpRfb) and ovary (AaLpRov) with respective molecular masses of 99.3 and 128.9 kDa. Each LpR variant encodes a member of the low density lipoprotein receptor family with five characteristic domains: 1) ligand recognition, 2) epidermal growth factor precursor, 3) putative O-linked sugar, 4) single membrane-spanning domains, and 5) the cytoplasmic tail with a highly conserved internalization signal FDNPVY. Proposed phylogenetic relationships among low density lipoprotein receptor superfamily members suggest that the LpRs of insects are more closely related to vertebrate low density lipoprotein receptors and very low density lipoprotein receptor/vitellogenin receptor than to insect vitellogenin receptor/yolk protein receptors. Two mosquito LpR isoforms differ in their amino termini, the ligand-binding domains, and O-linked sugar domains, which are generated by differential splicing. Polymerase chain reaction and Southern blot hybridization analyses show that these two transcripts originated from a single gene. Significantly, the putative ligand-binding domain consists of seven and eight complement-type, cysteine-rich repeats in AaLpRfb and AaLRov, respectively. Seven cysteine-rich repeats in AaLpRfb are identical to the second through eighth repeats of AaLpRov. Previous analyses (1) have indicated that the AaLpRov transcript is present exclusively in ovarian germ-line cells, nurse cells, and oocytes throughout the previtellogenic and vitellogenic stages, with the peak at 24-30 h after blood meal, coincident with the peak of yolk protein uptake. In contrast, the fat body-specific AaLpRfb transcript expression is restricted to the postvitellogenic period, during which yolk protein production is terminated and the fat body is transformed to a storage depot of lipid, carbohydrate, and protein.

The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

A simple and rapid method for cloning insect vitellogenin cDNAs

We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A) + RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7–0.9 kb, representing the 3′ portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene-specific primers corresponding to the sequences near the 5′-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5′ portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes.

Vitellogenin of Blattella germanica (L.) (Dictyoptera, blattellidae): nucleotide sequence of the cDNA and analysis of the protein primary structure.

The cloning and sequencing of a cDNA of the vitellogenin gene from the cockroach Blattella germanica is reported. It is 5,749 nucleotides long and encodes an amino acid sequence of 1,862 residues (including a putative signal peptide of 17 residues). The vitellogenin sequence includes a long serine-rich stretch between amino acids 322 and 349, and two other stretches between amino acids 1691 and 1740. The vitellogenin of B. germanica shows a notable similarity (between 32 and 42%) to those described in other insects, and its alignment shows a high number of motifs conserved in all species, especially in the subdomains I-V. Non-parsimony methods (Neighbor Joining) of phylogenetic analysis of the insect vitellogenin sequences gave a tree showing a topology that is, in general, congruent with the currently accepted insect phylogenetic schemes. Arch.

Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Atypical vitellins in ponerine ants (Formicidae: Hymenoptera)

Higher hymenopteran vitellogenin/vitellins have been characterized as containing one large apoprotein. We show that in the ant subfamily Ponerinae, species in the tribes Odontomachini, Platythyrini, and Amblyoponini, also have a vitellin with this simple structure, containing a single apoprotein of 180–190 kDa. Species in tribes Ponerini and Ectatommini, however, have vitellins containing multiple subunits. The size and number of the subunits varies, with differences even among species within the same genus. This is the first report of diversity in vitellogenin structure in the higher Hymenoptera. Vitellin and vitellogenin in Harpegnathos saltator (Ponerini) contain two large subunits of about 165 kDa and two small subunits of about 45 and 43 kDa. N-terminal sequence analysis suggests that provitellogenin is cleaved at two different sites, producing two large and two small subunits differing slightly in size. Diversity of vitellin types in Ponerini and Ectatommini is similar to that found in the more primitive tenthredinoid sawflies (Hymenoptera, Symphyta), and may indicate polyphyly in the Ponerinae. Insect vitellogenins and yolk proteins show considerably more diversity than originally believed, and the possibility of the functional significance of these differences should be considered.

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.

Extensive Sequence Conservation Among Insect, Nematode, and Vertebrate Vitellogenins Reveals Ancient Common Ancestry

The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect Vg sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence.

Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms

A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced ¶ ¶ Genbank accession number L41842. including 2015 bp of 5′ untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3′ untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C-terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis.