Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)

Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti‐80k vitellin antibody is cross‐reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and α‐chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph.     

Characterization of vitellin,egg-specific protein and 30 kDa protein from Bombyx eggs,and their fates during oogenesis and embryogenesis

Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

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.

     

Purification and partial characterization of vitellin from the eggs of the hard tick,Dermacentor variabilis

The major yolk proteins were purified from the eggs of the hard tick, Dermacentor variabilis using gel filtration and ion exchange chromatography. Two vitellin proteins were identified and designated vitellin A (480 kilodaltons; kDa) and vitellin B (370 kDa). The isolectric points were pH 6.1 and 6.25, respectively. The absorption maxima for both proteins were 280 and 400 nm. The buoyant density of vitellin A was 1.281 g/ml and vitellin B 1.278 g/ml. The vitellins were hemoglycolipoproteins as indicated by selective staining of polyacrylamide gels, carbohydrate analyses and lipid analyses. Under reducing conditions (SDS-PAGE), vitellin A had eight major polypeptides at 135, 110, 98, 80, 67, 50, 45, and 35 kDa. Vitellin B was identical to vitellin A with the addition of a 93 kDa subunit. The only carbohydrate detectable in the proteins was mannose. The neutral lipids detected in both proteins were cholesteryl esters, triglycerides, free fatty acids and their methyl esters, and cholestrol. The only detectable phospholipid in both proteins was phosphatidylethanolamine. The purified vitellins were immunologically identical to female hemolymph proteins but not to host hemoglobin. Antivitellin antibodies to vitellin were used to identify possible locations of vitellogenin in the organs of ovipositing females.     

红螯光壳螯虾胚胎及仔虾发育过程中卵黄磷蛋白的ELISA检测

建立了间接竞争酶联免疫吸附反应(ELISA)方法,对红螯光壳螯虾(Cherax quadricarinatus)胚胎及仔虾发育过程中的卵黄磷蛋白含量及其亚基组分进行了研究。该方法对卵黄磷蛋白具有良好的特异性,有效检测范围为31.25~250 ng/ml。结果表明,在发育初期胚胎先降解分子量比卵黄磷蛋白大的蛋白;亚基中,分子量较大和较小的亚基都先被消耗;胚胎内卵黄磷蛋白含量总体上呈下降趋势,其中在卵裂囊胚阶段后略有上升(3.19%),至后无节幼体期卵黄磷蛋白含量达最高(4.67%),之后不断下降,到仔虾离开母体独立生活时,含量只剩下卵裂期的1/4。     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。     

Purification and characterization of the common yolk protein,vitellin, from the estuarine amphipod Leptocheirus plumulosus

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.     

红褐斑腿蝗卵黄蛋白的分离纯化及性质分析

用蒸馏水沉淀法、凝胶过滤、蛋白质电泳等方法提取纯化了红褐斑腿蝗Catantops pinguis (Stål)的卵黄蛋白,并对其性质进行了分析。电泳结合不同的染色方法证明红褐斑腿蝗的卵黄蛋白为一种糖脂复合蛋白,其分子量约为548 kD,由7个亚基组成,亚基分子量分别为147.3,100.2,95.9,59.6,53.6,49.0和42.2 kD。卵黄蛋白经高效液相色谱分析检测到17种氨基酸和NH₃峰,其中谷氨酸（Glu）百分含量最高,达13.46%,天冬氨酸（Asp）、亮氨酸（Leu）、半胱氨酸（Cys）、精氨酸（Arg）含量比较高,脯氨酸（Pro）、组氨酸（His）、赖氨酸（Lys）含量较低。     

Effects of starvation on the vitellogenesis,ovarian development and fecundity in the ectoparasitoid,Nasonia vitripennis (Hymenoptera: Pteromalidae)

A female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.     

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476 ± 14.5 kDa as compared to 590 ± 25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

Isolation and characterization of vitellin from the fruitfly,Dacus oleae

Vitellin was isolated from mature eggs of Dacus oleae. A combination of anion-exchange chromatography and gel filtration was used for purification of the protein. The molecular weight of isolated vitellin, as determined by Sephacryl S-300 chromatography, was approximately 300,000. Electrophoresis on SDS-polyacrylamide gels demonstrated the presence of vitellin subunits with molecular weight of 47,000 and 49,000. Isoelectric focusing on polyacrylamide gels revealed a series of polypeptides with isoelectric points covering an acidic pH region of 5.7 to 6.2. Immunodiffusion, immunoelectrophoresis, and immunoblotting were used for further characterization of vitellin.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Jasmonic Acid and Salicylic Acid Induce Accumulation of β-1,3-Glucanase and Thaumatin-Like Proteins in Wheat and Enhance Resistance Against Stagonospora nodorum

The effect of application of jasmonic acid (JA) and salicylic acid (SA) on the induction of resistance in wheat to Stagonospora nodorum and on the induction of -1,3-glucanase and thaumatin-like proteins (TLPs) was studied. Western blot analysis revealed that two -1,3-glucanases with apparent molecular masses of 31 and 33 kDa that cross-reacted with a barley glucanase antiserum were induced in wheat leaves after treatment with JA and SA. When wheat plants were treated with SA and JA, a TLP with an apparent molecular mass of 25 kDa and several other isoforms of TLP were induced. Pre-treatment of wheat plants with SA and JA significantly reduced (up to 56 %) the incidence of leaf blotch disease incited by S. nodorum compared with untreated control plants.     

Vitellogenin sequestration by Simulium oocytes: the effect of Onchocerca infection

Abstract. The effects of Onchocerca infection on Simulium ornatum Meigen vitellogenesis were investigated. Vitellogenesis is triggered by ingestion of blood and is therefore linked to disease transmission. Vitellin subunits were described for the first time in the Simuliidae. In the British blackfiy S.ornatum , vitellin in the ovaries consists of two subunits, with a large subunit of molecular weight 200 kDa and a small subunit of 68 kDa. Simulium ornatum vitellin was recognized by polyclonal antibodies produced from Aedes aegypti (L.) vitellin. When blackflies were injected with Onchocerca lienalis (Stiles) microfilariae, there was a significant reduction in the amount of vitellin in the ovaries at 24, 34 and 50 h post blood-feeding.     

An LPS-binding hemagglutinin in the midgut of Triatoma infestans: Partial characterization and tissue localization

A hemagglutinin was identified and partially characterized in the crop of the bug Triatoma infestans. It agglutinated mouse and rabbit erythrocytes, and was strongly inhibited by bacterial lipopolysaccharides. SDS-PAGE and immunoblotting with specific antibodies identified a dominant protein with molecular mass of 20 kDa under reducing conditions. The protein seems to be synthesized in the crop epithelial cells and deposited on the inner crop surface. Its possible biological role is discussed. © 1995 Wiley-Liss, Inc.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

Sexual Dimorphism in Juvenile Hormone Synthesis by Corpora Allata and in Juvenile Hormone Acid Methyltransferase Activity in Corpora Allata and Accessory Sex Glands of Some Lepidoptera

Summary

The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E₂) on vitellin formation were examined in this study. The relative vitellin content measured by an enzyme-linked immunosorbent assay (ELISA) shows an apparent increase as the oocyte develops. Immunoblotting of the vitellin using anti-vitellin indicated that two main bands (179 and 110 kD), which begin to accumulate at an early stage of maturation, become pronounced during oocyte growth. Meanwhile, the major peak of the intact form of vitellin (530 kD) in gel filtration also enlarges with oocyte growth, supporting the results of immunoblot analysis and vitellin determination. E₂ treatment in vivo causes significant increases in oocyte diameter and vitellin content in the female oyster. A similar trend was observed in ovarian tissues cultured in the presence of E₂. It is concluded that E₂ is one of the major factors which control the vitellogenesis in the oyster and that the ovary is undoubtedly the site of synthesis of vitellin.