Archives of Insect Biochemistry and Physiology 6:39-48 (1987) Lipophorin in the Larval and Adult Stages of Musca dornestica Antonio G. de Bianchi, Margareth de L. Capurro, and Osvaldo Marinotti Departamento de Bioquimica, lnstituto de Quimica, Universidadede Sflo Paaulo, Brasil The major Mosca domestica hemolymph lipoprotein, lipophorin, was purified from larval and from adult animals. The housefly lipophorin is composed of two apoproteins, apolipophorin I (M, 253,000) and apolipophorin II (M, 85,000). The lipophorin contains about 3.9% carbohydrates and reacts positively with concanavalin A. The density of larval lipophorin is equal t o 1.152 glml and of adult lipophorin to 1.106 glml. The amount of lipophorin per animal increases during the larval stage, is constant during pupal stage, and suffers a great reduction at the pharate adult stage. The amount of lipophorin remains stable during the whole first gonotrophic cycle of the housefly. Lipophorin is not detected in the eggs of this insect. - - Key words: housefly, hemolymph proteins, metamorphosis INTRODUCTION Lipophorin, the major lipoprotein of insect hemolymph, is involved in lipid transport between the sites of absorption, storage, and utilization [1,2]. The lipophorin of several insects have been isolated and analyzed in the recent past [3-61. In general, the lipophorins are composed of two types of apoprotein, ApoLpI" and ApoLpII, with relative molecular weights of about 250,000 and 78,000, respectively . In some insects, a third apoprotein has been found in the adult lipophorin. This apoprotein, ApoLpIII, has a molecular weight of about 17,000 [6-81. *Abbreviations: ApoLp = apolipophorin; PAS = periodate-Schiff reagent; PTC = phenylthiocarbamide; SDS-PAGE = sodium dodecylsulfate polyacrylamidegel electrophoresis; TLCK = N-a-p-tosyl-L-lysinechloromethylketone. Acknowledgments: This work was supported by grants from the FundaC%ode Amparo B Pesquisa do Estado de SPo Paulo (FAPESP), Financiadora de Estudos e Projetos (FINEP), ConvCnio No. 43.84.0725.00. M.deL.C. i s a graduate fellow and O.M. a postdoctoral fellow of FAPESP. A.G.deB. is a staff member of the Biochemistry Department and a research fellow from CNPq. Received August 7, 1986; accepted March 16,1987. Address reprint requests to A.G. de Bianchi, Departamento de Bioquimica, lnstituto de Quimica, Universidade de S%oPaulo, C.P. 20780,01498, S%oPaulo, SP, Brasil. 0 1987 Alan R. Liss, Inc. 40 de Bianchi, de L. Capurro, and Marinotti The association of ApoLpIII with lipophorin is induced by the presence of adipokinetic hormone in the hemolymph, increasing the lipophorin lipidcarrying capacity [9,10]. ApoLpIII was found, until now, only in insects of the orders Lepidoptera, Hemiptera, and Orthoptera [6,7j. In addition to the ApoLpIII association with adult lipophorin, several stage-specific forms of this protein were recognized in Munducu sextu. These forms differ in their densities, and these differences are due to variation in lipid contents and composition [ll]. In adult Surcophugu bulZutu, the lipophorin is composed of ApoLpI and ApoLpII and has a density of 1.12g/ml. These are the only available data about lipophorin of dipteran insects. To obtain further information about dipteran lipophorins, we isolated and analyzed the Muscu dornestica lipophorin. In this paper, we also describe some aspects of qualitative and quantitative variations of this protein during the housefly life cycle. MATERIALS AND METHODS Animals M . dornesticu of a wild strain were reared at 25°C as described by Targa and Peres . Hemolymph, Fat Body, and Ovary Larval hemolymph was collected by puncturing the anterior end of the animals. The hemolymph was pooled in centrifuge tubes containing 0.1 M NaH2P04-NaOH,pH 7.0, 0.15 M NaC1, 5 mM EDTA, 1mM benzamidine, 0.5 mM PTC, and 1mM TLCK (buffer A). The diluted hemolymph (1:l)was centrifuged at 3,0008 for 10 min at 4"C, and the supernatant was used for the experiments. Adult hemolymph was obtained from C02-anesthetized flies. An abdominal incision was made in each fly with microscissors, and 5 pl of buffer A was used to wash out the hemolymph from the abdominal cavity. The diluted hemolymph was pooled in centrifuge tubes and centrifuged under the conditions described above. The ovaries and fat bodies were dissected out in 0.15 M NaC1. Isolation of Lipophorin Lipophorin was isolated by a modification of the density-gradient ultracentrifugation method described by Shapiro et al. . Hemolymph (2.3 ml) diluted in buffer A containing 3.7 M KBr was put into ultracentrifuge tubes and overlayed with 2.3 ml of buffer A. The tubes were centrifuged for 16 h at 96,0008 and 4°C in a Spinco SW50.1 rotor. The lipophorin, visible as a yellow band, was removed by inserting a syringe and withdrawing the contents. The density of lipophorin was determined from the refractive index of KBr of each sample at 25°C. Larval lipophorin isolated under these conditions is contaminated with low-molecular-weight polypeptides (see Results) and needs a glycerol-gradient ultracentrifugation step to separate it from these contaminants. A sample of partially purified lipophorin was laid on the top of a linear glycerol- Musca dornestica Lipophorin 41 gradient (5-30%) and centrifuged for 16 h at 96,OOOg at 4°C in a Spinco SW50.1 rotor. The lipophorin was collected as described above for the KBr gradients. Polyacrylamide Gel Electrophoresis SDS-PAGE was carried out according to the procedure of Laemmli . A 5-12% acrylamide gradient gel slab (9 cm) with a 1cm stacking gel was used. Electrophoresis was performed at a constant voltage of 7.8 Vlcm until the tracking dye reached the bottom of gels. Native electrophoresis was performed in 4% polyacrylamide gels according to the system described by Davis 1151. Gels were stained for protein with Coomassie blue R . The staining for glycoproteins was done with PAS [17I and for lipoproteins with Sudan black . Protein standards, for molecular weight determinations by the method of Lambin et al , were purchased from Sigma Chemical Co, (St. Louis, MO) and Bio-Rad Laboratories (Richmond, CA). Immunology About 0.6 mg of purified larval lipophorin was dissolved in 0.2 ml of 0.1 M NaH2P04-NaOHbuffer, pH 7.0, 0.15 M NaCl and emulsified with 0.5 ml of Freund’s complete adjuvant. This emulsion was injected subcutaneously, at multiple sites, into a rabbit. After 7 days, a similar injection was administered but with Freund’s incomplete adjuvant. Seven days after the last injection, the rabbit was bled and 0.01% (wh) NaN3 was added to the serum obtained before storage at -20°C. Double i ~ u n o d i f f u s i o nwas carried out according to the method of Ouchterlony  using 1%noble agar in 0.10 M NaH2P04-NaOHbuffer, pH 7.0, 0.3 M NaC1, 0.5 mM PTC, 5 mM EDTA, and 0.01% (wh) NaN3. Radial immunodiffusion  and rocket immunoelectrophoresis [Z]were used to estimate the lipophorin amount during the Musca life cycle. For radial immunodiffusion, samples of soluble proteins (see below), were applied to 3mm-diameter wells hollowed out in a 1-mm-thick slab of 1.5%noble agar in 0.1 M NaH2PO4-NaOH buffer, phf 7.0, 0.3 M NaCI, 0.05 m.M PTC, 0.01% (wfv) NaN3, and 1%(vh) antilipophorin serum. The rocket immunoelectrophoresis was run using 1.0% (wiv) agarose Type I (Sigma Chemical Co.) in 30 mM Tris, 83 mM sodium acetate, pH 8.6, O.OlO/c (wiv) NaN3. The serum antilipophorin was used at a concentration of 0.5%. The run was at 4.3 Vlcm for 16 h at room temperature. Quantificationof Lipophorin During Musca Life Cycle Animals were homogenized in buffer A (20 mg wet weight per 0.1 ml of buffer) in a Potter-Elvehjem homogeneizer. The homogenates were centrifuged at 11,OOOg for 10 min at 4°C. The supernatants (soluble proteins) were used for lipophorin quantification by radial immunodiffusion. Protein and Carbohydrate Determinations Protein was determined by the Coomassie blue method  using bovine serum albumin as standard. Carbohydrate was determined by the method of 42 de Bianchi, de 1. Capurro, and Marinotti Dubois et al.  using glucose as standard. To determine the contents of carbohydrate of purified lipophorin, protein was estimated by the method of Ellman . RESULTS Lipophorin from the hemolymph of adults and larva of M. domestica was isolated as described in Materials and Methods. The purity of the lipophorin preparations was assessed by SDS-PAGE (Fig. 1A). After the KBr densitygradient ultracentrifugation, the larval Iipophorin is contaminated with lowmolecular-weight polypeptides (Fig. 1A) that are removed by the glycerolgradient ultracentrifugation step (Fig. 1A). The adult lipophorin obtained is free of contaminants after the one-step KBr density-gradient ultracentrifugation (Fig. 1A). The purified larval lipophorin of M. domestica, when analyzed by native electrophoresis, gives a very well defined band that is stained by PAS and Sudan black (Fig. 1B). Under these conditions, the lipophorin shows some aggregation as evidenced by the protein band retained at the top of the running gel (Fig. 1B). When this retained band is cut out of the polyacrylamide cylinder and analyzed by SDS-PAGE, only lipophorin subunits are detected (results not shown). The M. dumestica lipophorin (from adult and larva) is composed of two types of apoprotein, ApoLpI and ApoLpII, with relative molecular weights of 253,000 f 6,000 and 85,000 f 3,000, respectively (Fig. 2). The subunits are dissociated by 0.1% (wh) SDS. The purified lipophorin has about 3.9% B A 1 2 3 1 2 3 Fig. 1. A SDS-PACE (5-12% acrylamide gradient slab) of Musca domestica lipophorin. Lane 1: Purified larval lipophorin after KBr gradient centrifugation. Lane 2: Purified larval lipophorin after glycerol gradient centrifugation. Lane 3: Purified adult lipophorin after KBr gradient centrifugation. B: Native electrophoresis of purified larval M. domesfica lipophorin in 4% polyacrylamide gels. Lane 1: Coomassie blue R staining. Lane 2: PAS staining. Lane 3: Sudan black staining. Musca domestica Lipophorin 43 0 4.0- Log x T Fig. 2. Determination of the molecular weights of apolipophorin I and apolipophorin I I from larval M. domestica lipophorin. The proteins were subjected to SDS-PAGE (5-12% acrylamide gradient slab), and the molecular weights were estimated by the method of Lambin et al. . The arrows indicate the molecular weights of ApoLpl and ApoLpll. The proteins used as molecular weight markers were 1, thyroglobulin; 2, myosin; 3, P-galactosidase; 4, phosphorilase B; 5, bovine serum albumin; 6, ovalbumin; 7, carbonic anhydrase; and 8, soybean trypsin inhibitor. The straight line was constructed using the least-squares method. The correlation coefficient (r) was equal to 0.999. 0.7% of carbohydrates, and, since lipophorin reacts positively with concanavalin A (Fig. 3C), glucose andlor mannose must be associated with this protein. The densities of lipophorins of the several developmental stages of M . domeslim are given in Table 1. The adult lipophorin shows a significantly lower density than the lipophorin at other stages. Immunology The serum antilipophorin is monospecific when tested by double immunodiffusion against larval and adult hemolymph (Fig. 3). In spite of the difference in density values, the adult and larval lipophorins are immunologically identical (Fig. 3). The double immunodiffusion tests were unable to demonstrate lipophorin presence in fat bodies, ovaries, and eggs (Fig. 3). Analysis by rocket immunoelectrophoresis indicates that lipophorin, if present, must represent less than 0.02% of total egg protein (Fig. 4). 44 de Bianchi, de L, Capurro, and Marinotti C B A Fig. 3. A Ouchterlony immunodiffusion in 1%noble agar. Central well contained antilipophorin serum and the others contained 1 and 4, purified larval lipophorin; 2, adult hemalymph; and 3, larval hemolymph. B Ouchterlony immunodiffusion in 1%noble agar. Central well contained a n t i l i ~ ~ h o serum ~ i n and the others contained 1 and 4, purified larval lipophorin; 2, egg soluble proteins: 3, soluble ovarian proteins; 5, soluble larval fat body proteins; and 6, soluble adult fat body proteins. C Double diffusion of concanavalin A against purified larval M. domestica lipophorin. Central well contained 20 pg of concanavalin A, and wells 1, 2, 3, 4,5 and 6 contained 20, 10,5,2,1, and 0.5 p g of purified lipophorin, respectively. TABLE 1. Lipophorin Density Values During Musca domestica Life Cycle Density (gimlfa Developmental stages Larval (feeding) Larval (wandering) Pupal Adult 1.152 f 0.004 1.145 f 0.003 1.142 f 0.002 1.106 f 0.007 “Values are mean f SD (n = 3). Lipophorin Quantification The growth of M. ~ o ~ ebys wet t ~weight ~ under our rearing conditions is shown in Figure 5. After the beginning of the adult stage, the female flies grow larger than males because of ovarian development. The quantification by radial immunodiffusion of lipophorin in houseflies during their life cycle is presented in Figure 6. The quantity of lipophorin increases during the larval stage, remains at a high and relatively stable level during all the pupal stage, and suffers a great reduction at the pharate adult stage. The lipophorin level is stable during the entire first gonotrophic cycle of the housefly. The reduction in lipophorin level at the pharate adult stage is not due to a reduction in weight of the flies, which is stable in the pupaladult transition (Fig. 5). DISCUSSION The M . domesticrz lipophorin is composed of two types of apolipoproteins, ApoLpI (N, 253,000) and ApoLPII (M, 85,000). The molecular weights of the apolipophorins are reported to be about 250,000 for ApoLpI and 78,000 for ApoLpII across a wide spectrum of insect orders . Some insects in the adult stage show a lipophorin formed by three apoproteins ; in others, the lipophorin is assembled only from two apolipoproteins, as is the case for housefly lipophorin. - - Musca dornestica Lipophorin 1 larva I pupa 45 ad u lt 1I i 5 5 10 ii Age in days Fig. 4. Rocket immunoelectrophoresis of lipophorin. Lipophorin was quantitated by rocket immunoelectrophoresis as described in Materials and Methods. Samples of recently formed pupa homogenates were applied to wells 1, 2, and 3. The samples contained 0.60, 0.56, and 0.68 pg of lipophorin, respectively. Well 4 contained egg homogenate (94 p g of total soluble protein). Fig. 5. M. domestica wet weight during development. Means & SD of three determinations on groups of five to 20 animals. The age in days after egg laying i s indicated as are the larval, pupal, and adult stages. S4, Sbr and S8 indicate the ovarian stages during the first gonotrophic cycle according to Adams 1321. After emergence, the values for females ( 0 - - - - 0 )and males (0-0) are individualized. 46 de Bianchi, de 1. Capurro, and Marinotti I larva I pupa adult 20- 5 5 10 15 Age in d a y s Fig. 6. Total lipophorin in M. dornestica during development. Each point is the mean k SD of three determinations on groups of five to 20 animals. Lipophorin was quantitated by radial immunodiffusion as described in Materials and Methods. The developmental stages are indicated at the top. S4, S6, S,, and S,, indicate the ovarian stages during the first gonotrophic and males cycle according to Adams .After emergence, the values for females -I(. (0-0) are individualized. The carbohydrate contents of housefly lipophorin (3.9%) are similar to those described for Locustu rnigrutoria . Covalently bound mannose-containing oligosaccharide chains are reported to occur in the lipophorin of several insect orders 161 as demonstrated by positive reaction of lipophorin with concanavalin A. We also reported the presence of this type of oligosaccharide chain in lipophorin from M . dornesticu larvae. The densities of housefly lipophorin suggest that, during the feeding, wandering, and pupal stages, the lipoprotein does not undergo major modifications in its lipid composition. On the other hand, the adult lipophorin shows a lower density, suggesting a qualitative andlor quantitative modification in the lipids carried by the protein. Modifications in the densities of Munducu sextu lipophorin during the life cycle of this insect were reported by Sarvamangala et al. [ll]. The alteration of Munducu sextu lipophorin density values were correlated with quantitative and qualitative modifications of the lipid core of the protein [ll].The several forms of Munducu sextu lipophorin are interconvertible by action of a lipid transfer protein [26,27]. The density of housefly lipophorin (1.106 glml) is lower than that reported for Surcophugu bullutu (1.12 glml) , the only density value for adult dipteran lipophorin reported until now. The quantification of lipophorin during the life cycle of the housefly shows very interesting features. The results (Fig. 6) suggest that lipophorin is synthesked in very significant amounts only during the larval stage. During the pupal stage, the synthesis of lipophorin is probably at a low level, because the amount of the protein is stable (Fig. 6), and the turnover of Musca domestica Lipophorin 47 lipophorin is known to be very slow in other insects . The very wellmarked time of reduction in the lipophorin level suggests a well-tuned mechanism for protein degradation that is activated in the pharate adult stage. The level of M. dornesficu storage protein is reduced at precisely the same time as lipophorin 1291. 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