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.     

Purification and properties of a very high density lipoprotein from the hemolymph of the honeybee Apis mellifera

A larval-specific very high density lipoprotein (VHDL) has been isolated from the hemolymph of the honeybee Apis mellifera. VHDL was isolated by a combination of density gradient ultracentrifugation and gel filtration. The purified protein is a dimer of Mr 160,000 apoproteins as shown by chemical cross-linking with dimethyl suberimidate. N-Terminal sequence analysis indicates that the two polypeptide chains are identical. The holoprotein contains 10% lipid by weight and 2.6% covalently bound carbohydrate. A native Mr 330,000 species was obtained by gel permeation chromatography. Antiserum directed against VHDL was used to show that VHDL is distinct from other hemolymph proteins and appears to constitute a novel lipoprotein of unknown function. However, the lipoprotein is present in high amounts in hemolymph only at the end of larval life, suggesting a potential role in lipid transport and/or storage protein metabolism during metamorphosis.     

Vitellogenin Recognizes Cell Damage through Membrane Binding and Shields Living Cells from Reactive Oxygen Species

Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span.     

Adaptive evolution of a key gene affecting queen and worker traits in the honey bee, Apis mellifera

The vitellogenin egg yolk precursor protein represents a well-studied case of social pleiotropy in the model organism Apis mellifera. Vitellogenin is associated with fecundity in queens and plays a major role in controlling division of labour in workers, thereby affecting both individual and colony-level fitness. We studied the molecular evolution of vitellogenin and seven other genes sequenced in a large population panel of Apis mellifera and several closely related species to investigate the role of social pleiotropy on adaptive protein evolution. We found a significant excess of nonsynonymous fixed differences between A. mellifera, A. cerana and A. florea relative to synonymous sites indicating high rates of adaptive evolution at vitellogenin. Indeed, 88% of amino acid changes were fixed by selection in some portions of the gene. Further, vitellogenin exhibited hallmark signatures of selective sweeps in A. mellifera, including a significant skew in the allele frequency spectrum, extreme levels of genetic differentiation and linkage disequilibrium. Finally, replacement polymorphisms in vitellogenin were significantly enriched in parts of the protein involved in binding lipid, establishing a link between the gene's structure, function and effects on fitness. Our case study provides unequivocal evidence of historical and ongoing bouts of adaptive evolution acting on a key socially pleiotropic gene in the honey bee.     

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

Osmia 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.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Getting a buzz out of the bee genome

The honey bee Apis mellifera displays the most complex behavior of any insect. This, and its utility to humans, makes it a fascinating object of study for biologists. Such studies are now further enabled by the release of the honey-bee genome sequence.     

昆虫卵黄原蛋白功能多效性：以蜜蜂为例

卵黄原蛋白是昆虫卵黄发生的关键物质。随着RNA干扰技术在功能基因研究中的应用和发展,昆虫卵黄原蛋白特别是蜜蜂卵黄原蛋白被发现具有气候适应、激活卵巢、生殖竞争、劳动力分化、行为构建、延长寿命、转化食物等多种功能,因此又被称为多效性蛋白。许多针对蜜蜂卵黄原蛋白功能和调控机制的假说也相继提出,如“交互抑制假说”、“循环反馈机制”、“卵黄原蛋白转化蛋白胨机制”,表明蜜蜂卵黄原蛋白已经由雌虫繁殖过程的一个下游因子上升为高等社会性昆虫主要生命周期的调控子,不仅极大地促进了昆虫社会生物学的研究,对其他具有复杂繁殖行为的昆虫的研究也具有重要的参考意义。针对近年来这一领域相继取得的重大突破,本文以蜜蜂为例介绍了昆虫卵黄原蛋白功能多效性的研究进展,包括卵黄原蛋白的理化性质与分子进化,发生与调控,功能多效性及其研究方法等     

Homology of Drosophila yolk proteins and the triacylglycerol lipase family

Drosophila yolk proteins consist of a set of related proteins of 50,000 Mr. They are derived from slightly larger precursors by cleavage of a signal peptide. In this respect, they differ from the yolk proteins of other insects which are proteolytic fragments of precursors of 200,000 Mr or larger, termed vitellogenins and probably homologous to the vitellogenins of other egg-laying species. We report here a comparative amino acid analysis demonstrating that the Drosophila yolk proteins are non-homologous to the vitellogenin group of yolk proteins, but surprisingly are related to the triacylglycerol lipase family.     

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.     

Introgression in native populations of <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> L: implications for conservation

Hybridisation and introgression can have negative impacts on regional biodiversity through the potential erosion of locally adapted lineages. The honey bee (Apis mellifera L.) occurs in twenty-seven subspecies across Europe, is an extremely economically important insect, yet threatened by multifarious impacts. Transhumance of the most commercially appealing varieties threatens native honey bee diversity by introgression and subsequent loss of locally adapted traits, or even by complete removal of some subspecies from parts of the range. Here levels of admixture and introgression are examined in UK honey bees suspected to be from hives of the dark European honey bee (Apis mellifera mellifera). Microsatellite DNA and STRUCTURE analyses reveal that the studied populations are generally admixed, and discriminant analysis of principal components shows them to be intermediate between A. m. mellifera and Apis mellifera carnica populations. However, examining mitochondrial haplotype data (COI-COII intergenic spacer region) and nuclear DNA reveal that some hives are relatively pure (from 4 to 15 hives, depending on the Q-value threshold). Genetic diversity is relatively high in comparison with other European populations. Implications for conservation and management are discussed.     

Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee,Apis cerana japonica (Hymenoptera,Apocrita)

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.     

Bees brought to their knees: microbes affecting honey bee health

The biology and health of the honey bee Apis mellifera has been of interest to human societies for centuries. Research on honey bee health is surging, in part due to new tools and the arrival of colony-collapse disorder (CCD), an unsolved decline in bees from parts of the United States, Europe, and Asia. Although a clear understanding of what causes CCD has yet to emerge, these efforts have led to new microbial discoveries and avenues to improve our understanding of bees and the challenges they face. Here we review the known honey bee microbes and highlight areas of both active and lagging research. Detailed studies of honey bee-pathogen dynamics will help efforts to keep this important pollinator healthy and will give general insights into both beneficial and harmful microbes confronting insect colonies.     

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.     

VHDL, a larval storage protein from the corn earworm, Helicoverpa zea, is a member of the vitellogenin gene family

The hemolymph of last instar larvae of the corn earworm, Helicoverpa zea contains a blue very high-density lipoprotein (VHDL) that is selectively taken up into fat body prior to pupation. Its amino-terminal sequence was determined by Edman degradation, and used to design a degenerate primer for PCR amplification. With 5' and 3' RACE techniques, the entire cDNA coding for VHDL was amplified and sequenced. Conceptual translation reveals a 173 kDa protein that contains a 15 amino acid signal sequence immediately before the experimentally determined N-terminus of the mature protein. The protein contains a typical lipoprotein N-terminal domain, and shows high sequence similarity to vitellogenins from Lepidoptera and other insect species. VHDL mRNA was not detectable in adult H. zea, and antibodies raised against VHDL did not react with adult hemolymph or yolk proteins. Therefore VHDL, although a member of the vitellogenin gene family, seems to be distinct from the vitellogenin expressed in adult females.     

Molecular identification of the first SIFamide receptor

SIFamide is the short name and also the C terminus of the Drosophila neuropeptide AYRKPPFNGSIFamide. SIFamide has been isolated or predicted from various insects and crustaceans, and appears to be extremely well conserved among these arthropods. However, the function of this neuropeptide is still enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines. Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor.