Influence of the blood meal source on vitellogenin synthesis by isolated fat bodies of Rhodnius prolixus

Insect Biochem. M&c. Bid. Vol. 22, No. 2, pp. 159-166,1992 Printed in Great Britain. All rights reserved

INFLUENCE VITELLOGENIN

Departamento

0965-1748/92$5.00+ 0.00 Copyright 0 1992Pergamon Press plc

OF THE BLOOD MEAL SOURCE ON SYNTHESIS BY ISOLATED FAT BODIES OF RHODNIUS PROLIXUS

DENISE VALLE, E. S. GARCIA and S. GOLDENBERG* de Bioquimica e Biologia Molecular, Funda@o Oswald0 Cruz, Av. Brasil 4365,

Rio de Janeiro 21040, Brasil (Received 9 May 1991; revised and accepted 12 September 1991)

Abstract-In

an attempt to study the kinetics of vitellogenin (Vg) synthesis in the triatomidae Rhodnius

prolixus,

isolated fat bodies from adult females were metabolically labelled with [%]methionine and the proteins were analysed by SDS-PAGE after immunoprecipitation with an anti-egg serum. It was observed that the kinetics of Vg synthesis varies depending on the blood source used to feed the insects. Furthermore, the secondary cycle of Vg synthesis occurs faster than the first one. It was also shown that the various Vg subunits are differentially expressed after feeding. Additionally, polypeptides of 205 and 190 kDa were detected that display synthesis kinetics similar to Vg, are not secreted to the incubation medium and are immunoprecipitable by an anti-Vg specific serum. These polypeptides could be potential precursors of Vg subunits. Key Word Index: fat body; Rhodnius prolixus; vitellogenin; vitellogenin synthesis

INTRODUCTION

Vitellogenesis is a rapid process of oocyte development resulting in the accumulation of vitellus, of which the protein vitellin (Vn) is the major componnent (Pan et al., 1969; Postlethwait and Giorgi, 1985). This oocyte accumulation of Vn is accompanied by a massive synthesis of its haemolymphatic homologue, the protein vitellogenin (Vg). The fat body is the exclusive site of Vg synthesis for the majority of insects, with the exception of some Diptera, where some Vg synthesis occurs in the ovaries (Bownes, 1980; Zongza and Dimitridiadis, 1988; Peferoen and DeLoof, 1986). There is evidence, in both insects and vertebrates, that Vg synthesis occurs faster during the second cycle of oviposition or hormonal stimulation than during the first one (Tata and Smith, 1979; Kunkel and Nordin, 1984). This has been attributed to a requirement for cytological preparation of the fat body, known as the pre-vitellogenic phase (Raikhel and Lea, 1983). In mosquitoes, egg production levels have been related to the blood feeding source (Woke, 1937; Shelton, 1973). For example, in Aedes uegypti this phenomenon was found to be related to the isoleucine levels in the blood used to feed the insects (Chang and Judson, 1977). Previous work in our laboratory has also shown an influence of the blood source on several aspects of Abbreviations used: FBL: Fat body lysate, IM: incubation

medium, mol. wt: molecular weight, PMSF: phenylmethyl sulphonyl fluoride, SDS-PAGE, polyacrylamide gel electrophoresis with sodium dodecyl sulfate, TLCK: tosyl lysyl chloromethyl ketone, Vg: vitellogenin, Vn: vitellin.

Rhodnius prolixus metabolism (Lima Gomes, 1986; Lima Gomes et al., 1990). It has also been shown that the blood source can influence egg production levels and autogeny in R. prolixus (Valle et al., 1987). In this case isoleucine does not seem to have any influence, since human blood, which is poor in this amino acid, is the feeding source that yields the best results in terms of development and egg production in this insect (Lima Gomes et al., 1990). We have shown that the pattern obtained upon feeding on human blood (autogenic and first cycle number of eggs) can also be obtained when a colony fed on sheep blood receives human blood from the 4th instar onwards (Valle et al., 1987). In the present work we compare Vg synthesis kinetics of two differently fed R. prolixus colonies: one received exclusively human blood and a second colony was fed on sheep blood until 3rd instar and then on human blood, from 4th instar onwards. The objective of the comparison was to see if the pattern of the human blood-fed colony, with respect to Vg synthesis by the fat bodies, was also obtained by the second colony. In this later colony we also compare differential Vg synthesis kinetics during the first and second oviposition cycles.

MATERIALS AND METHODS Insects R. prolixus colonies were maintained at 28°C in 70% r.h. and were artificially fed (Garcia et al., 1984). Two colonies of adult insects were used: one fed exclusively on human blood (55.5 f 6.9 mg, n = 40) and the other fed on sheep blood until the 3rd instar, and on human blood from the 4th instar onwards (42.1 f 4.4 mg, n = 40). Only fully gorged insects were used (188.4 f 13.5 mg after feeding, n = 30); partially fed animals were rejected.

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DENISE VALLE et al

A R. prolixus colony fed exclusively on sheep blood was not included because individual variations in terms of status of vitellogenesis as a function of time (size of ovaries and aspect of fat bodies) were so extensive that results were not conclusive.

150 kDa and two in the range of 50 kDa (Masuda and Oliveira, 1985). Accordingly, our previous work resulted in the detection of four polypeptides as components of R. prolixus Vg, with molecular weights of 159, 148, 53 and 48 kDa (Valle et al., 1987). Protein synthesis by isolated fat bodies In R. prolixus the blood meal triggers all the major At each timepoint, abdominal fat bodies from four insects metabolic events (Buxton, 1930), including Vg synwere dissected, rinsed in 100 mM NaCl, 25 mM KC1 and thesis. In order to study the kinetics of this process, IOmM CaC12 and pooled in incubation medium (MEM, GIBCO, depleted of methionine) buffered with 15mM isolated abdominal fat bodies from adult females HEPES buffer (N-2-hydroxyethylpiperazine-N’-2-ethanewere metabolically labelled with [35S]methionine as sulfonic acid) pH 7.0. Fat bodies were pre-incubated with described in Materials and Methods. It is worth gentle agitation for 30min at 28°C in 1 ml of medium mentioning that the samples used for analysis were and then, incubated at 28°C with 20 &i of [35S]methionine not standardized according to protein content, since (13OOCi/mmol, Amersham International) in a total volume this varies greatly during the time period studied: of 400 ~1, for 2 h. After this time the incubation medium (IM) was separated and centrifuged twice at 12,OOOg we used equivalent numbers of fat bodies (i.e. for each time point, the same numbers of fat bodies were for 5 min to remove debris. The pooled fat bodies were extracted into a constant volume, as described in homoeenized in 400 ~1 of lvsis buffer (10 mM NaCl. 5 mM MgCI:, 50 mM Tris’-HCl pH 7.6 and 1% NP-40). After Materials and Methods). In addition, the fat body two centrifugations of 5 min at 12,OOOg,the supernatant lysate (FBL) and the incubation media (IM) were was used as the fat body lysate (FBL). Proteinase inhibitors analysed separately, to allow the observation of 2mM each of PMSF (phenylmethyl sulphonydil fluoride) proteins synthesized and secreted by the fat bodies, and TLCK (tosyl lysyl chloromethyl ketone) were added respectively. to the IM before centrifugations and to the FBL before Fat bodies from first cycle female adults fed exclushomogenization. ively on human blood were subjected to [3SS]methAntisera and immunoprecipitations ionine incorporation at different days after feeding. Figure l(A) shows the time course of immunoFor production of rabbit antiserum against R. prolixus oocytes, two subcutaneous injections of 2 mg of egg extract precipitation with “anti-egg” serum of the synthesized protein in Freund’s adjuvant (complete and incomplete, proteins. It can be observed that Vg synthesis reached successively) were given, with a 6 weeks interval between a plateau 2 days after feeding. SDS-PAGE analysis injections. Six weeks after the second subcutaneous injecof the immunoprecipitated proteins synthesized and tion, an intravenous booster was given, with 500 pg of egg secreted are presented in Fig. 1, panels B and C, extract. The animals were bled 10 days later. This “anti-egg” respectively. After the [35S]methionine incorporation serum recognizes, in addition to Vg, polypeptides in the the 53 and 48 kDa Vg subunits are clearly visible only 70-80 kDa range, which are abundant in the haemolymph with an overexposure of the gels [Fig l(D)]. Because and seem to be related to storage proteins (Rehn and Rolim, this procedure prejudices the visualization of the 1990). This antiserum was used in all the immunoptecipitations performed with the exception of the one shown in Fig. higher mol. wt polypeptides, we decided to use shorter 2, where we used anti-Vg specific serum. This latter serum exposure times and to concentrate the analysis on was obtained by extensive immunoabsorption of “anti-egg” the higher mol. wt polypeptides. The Vg subunit of serum with 5th instar haemolymphatic proteins immobilized 159 kDa could be observed 5 h after feeding [day 0 on on CNBr activated Sepharose (Pharmacia), according to the Fig. l(B), (C)l. On the other hand, the subunit of manufacturer’s protocol. 5th instar haemolymphatic pro148 kDa could be observed only on the second day. teins have previously been shown to contain the 70-80 kDa However, the rate of synthesis of this subunit was at polypeptides, but not Vg subunits. The immunoprecipitations were performed essentially as its maximum by that time, since the pattern did not described by Contreras et al. (1985), using an excess of the change between the second and fourth day [Fig. l(B)]. specific antiserum. Prior to analysis by electrophoresis the The “anti-egg” serum also recognized two polyradioactivity of aliquots of the immunoprecipitated proteins peptides of 205 and 190 kDa in the FBL. The kinetics was determined. of synthesis of these polypeptides accompanied that of Vg, although they were not secreted into the IM Protein gel electrophoresis [Fig. l(C)]. Samples were analyzed by SDS-PAGE, as described by Since the “anti-egg” serum used in this study is not Laemmli (1970). The molecular sizes of the polypeptides monospecific, the kinetics of synthesis and secretion were determined by comparison with the migration of comof the 70-80 kDa polypeptides can also be observed mercial “C-protein molecular weight (mol. wt) standards (Fig. 1). Their kinetics in adult females roughly (BRL). After electrophoresis the gels were fixed, treated for fluorography (Laskey and Mills, 1975), dried and exposed parallels that of Vg in this colony of human blood fed to Kodak X-AR films at - 70°C with Kodak X-O-MAT insects. intensifying screens. In order to investigate whether the 205, 190 and 70-80 kDa polypeptides are structurally related to Vg, the anti-egg serum was rendered Vg specific (see RESULTS AND DISCUSSION Materials and Methods) and used to immunoprecipiR. prolixus Vg and Vn have been previously tate the FBL sample from the 4th day after feeding. characterized as phospholipoglycoproteins present in The immunoprecipitation products were analysed by two different native aggregational states of distinct SDS-PAGE and the result is shown in Fig. 2. It can mol. wt, as seen in non-denaturing gels. Each of these be observed that the 70-80 kDa polypeptides are not aggregates has been shown, when subjected to deimmunoprecipitated by the anti-Vg specific serum. naturing conditions, to release the same 4 polypepNevertheless, the 205 and 190 kDa polypeptides seem tides, two of approx.

4 (A)

3

P y

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(D)

(C)

(B) 0

1

2

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200<159 Cl48
159 148

9768-

c53 C48 43-

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Fig. 1. Kinetics of [35S]methionine incorporation by isolated fat bodies of first cycle female adults from a colony fed exclusively on human blood. (A) Time course of labelled proteins from the fat body lysate of a pool of 4 females immunoprecipitated with anti-egg serum. (B)-(D) SDS-PAGE analysis of proteins immunoprecipitated with anti-egg serum. (B) and (D) fat body lysate; (C) incubation medium. Numbers above the lanes indicate the days when the incorporation was done. Day 0 means 5 h after feeding. Panel D is an overexposure of panel B, 4 days after feeding. The position of commercial mol. wt markers are indicated in the left of each panel and the sizes of Vg subunits are shown in the right. The arrowheads in A indicate the higher mol. wt precursor Vg polypeptides described in the text.

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Fig. 2. Analysis by SDS-PAGE of immunoprecipitated polypeptides of the fat body lysate on the fourth day after feeding with anti-VP specific serum. Legends as in Fig. 1.

5 (A)

4

1

3

< r x2 B 1

0 1

0

4

3 -2-W

(C)

(B) 0

1

2

3

4

coo zoo-

97

159 146

97 -

686643 43-

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Fig. 3. Kinetics of [35S]methionine incorporation by isolated fat bodies of female adults from a colony fed exclusively on sheep blood until third instar and on human blood from fourth instar on. (A) Time course of labelled proteins from the fat body lysate of a pool of 4 females immunoprecipitated with anti-egg serum. Analysis of(e) first oviposition cycle females and (m) second cycle females. (B) and (C) SDS-PAGE analysis of labelled proteins immunoprecipitated with anti-egg serum. Fat bodies from females in the second cycle of oviposition (B) or in the first cycle of oviposition (C). Legends as in Fig. 1.

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Influence of the blood meal source on vitellogenin synthesis to have a structural

homology to Vg, since they were immunoprecipitated by the serum. Taken together these results indicate that: (1) for first vitellogenic cycle females fed on human blood, maximum Vg synthesis is attained 2 days after feeding; (2) the various Vg subunits appear differentially in time and (3) polypeptides of 205 and 190 kDa are synthesized concomitantly with the Vg subunits, they remain inside the fat bodies and they are immunologically related to Vg subunits (Fig. 2). Whether they are derived from different genes or are the result of differential processing of the same gene remains to be elucidated. Vg precursors of higher mol. wt have been detected in other insects such as Hyalophora cecropia, Tenebrio molitor (Harnish and White, 1982), Leucophaea maderae (Koeppe and Ofengand, 1976; Della-Cloppa and Engelmann, 1987), Locusta migratoria (Chen et al., 1978), Periplaneta americana (Sams et al., 1980) and Bacillus rossius (Giorgi et al., 1989). Previous data from our laboratory have shown that the oviposition pattern of a R. prolixus colony fed on human blood could be almost completely restored in a sheep blood fed colony that was given human blood from the 4th instar onwards (Valle et al., 1987). We decided to analyse the kinetics of Vg synthesis from this latter group in order to see if the “human blood pattern” was also restored with respect to this parameter. The results are shown on Fig. 3, where isolated fat bodies from adult females from a colony fed on human blood since the 4th instar (and on sheep blood beforehand) were labelled with [‘%]methionine and the proteins immunoprecipitated with “anti-egg” serum, exactly as for Fig. 1. Only the FBL samples are shown. Again the samples were normalized by fat body number equivalents. In this colony the maximum level of Vg synthesis was not reached on the second day after feeding. The kinetics shown in Fig. 3(A) shows that for first cycle females Vg synthesis is still increasing until the 4th day after feeding. Additionally, comparison of the different lanes of Fig. 3(C) shows an increasing rate of synthesis throughout the course of the experiment, as judged by the relative intensities of the protein bands. Furthermore it could again be observed that the polypeptides of 205 and 190 kDa accompanied Vg kinetics. A differential timing of appearance of the various Vg subunits could still be observed, in agreement with the results obtained with the human blood fed females (Fig. 1). The Vg polypeptide of 159 kDa was detectable 5 h after feeding [day 0 on Fig. 3(C)] whereas the 148 kDa Vg subunit could be clearly observed only from the third day onwards. Figure 3(C) also permits visualization of the 70-80 kDa polypeptides, and shows that maximum levels of synthesis are achieved at the third day. This corroborates our previous observation (Fig. 2) that this group is not related to vg. The results indicate that at the level of Vg synthesis, feeding on human blood from the 4th instar onwards is not sufficient to completely revert the Vg synthesis pattern of a sheep blood-fed colony to that of the colony fed exclusively on human blood. In this latter colony first cycle Vg synthesis reaches its maximum rate 2 days after feeding (Fig. 1) while in the former

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the intensity of Vg synthesis is still increasing on the fourth day after feeding [Fig. 3(A)], indicating a slower stimulation. Synthesis of Vg and oviposition can be seen as different levels of expression of Vg genes. Because Vg synthesis represents an earlier event than oviposition in the cascade of events that lead to the general phenomenon of vitellogenesis, it may reflect the influence of parameters that alter Vg genes expression more sensitively. Other parameters, besides Vg synthesis, have to be taken into account for oviposition to be accomplished, namely processing of Vg precursors, assembly of the protein, secretion to the haemolymph and uptake by the oocytes. For this reason we believe that greater differences can be seen between differently fed colonies when we analyse Vg synthesis instead of oviposition. The second cycle of vitellogenesis occurs faster than the first one (Chinzei et al., 1982; Kunkel and Nordin, 1984; Tata and Smith, 1979). We used the approach described above to test the Vg synthesis kinetics of second cycle adult females from the colony fed on human blood from the 4th instar onwards. The results are presented on Fig. 3(A) where it can be seen that the second cycle initial stimulation of Vg synthesis is faster than the first one. Another difference between both cycles concerns the timing of synthesis of the 148 kDa polypeptide; in the second cycle it can be detected from day 1 on [Fig. 3(B)] whereas it is detectable only after day 3 of feeding in the first cycle [Fig. 3(C)]. It is possible that cytoplasmic regulation of Vg mRNA expression might play a role in this process. Indeed, it has been observed in L. migratoria that at the end of primary stimulation the level of Vg decreases much more rapidly than that of Vg mRNA levels (Chinxei et al., 1982). Hence, it is possible that in R. prolixus the Vg mRNA is already present at the end of the primary stimulation, and that it is available for translation again only after the secondary cycle. Acknowledgements-We thank Dr Flavio S. Faria for helpful discussions, Dr Catherine Lowndes and Dr Walter Geleman for the critical reading of this manuscript. This work received financial support from Financiadora de Estudos e Projetos (FINEP), Con&ho National de Desenvolvimento Cientifico e Tecnologico (CNPq) and FAPERJ (Fundacgo de Amparo a Pesquisa do Estado do Rio de Janeiro).

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