- Email: [email protected]
Physiological pattern and further characterization of the haemolymph violet carotenoprotein of the fly Rhynchosciara americana
Comp. Biochem. Physiol. Vol. 83B, No. 3, pp. 637 642, 1986 Printed in Great Britain
0305-0491/86 $3,00+0.00 (C) 1986 Pergamon Press Ltd
PHYSIOLOGICAL PATTERN A N D F U R T H E R C H A R A C T E R I Z A T I O N OF T H E H A E M O L Y M P H V I O L E T C A R O T E N O P R O T E I N OF T H E F L Y RHYNCHOSCIARA AMERICANA A. G. DE OlANCIII and O. MARINOTTI Departamento de Bioquimica, Instituto de Quimica, Universidade de S~o Paulo, SP., C.P. 20780, CEP 01498--S~o Paulo, Brasil (Tel: 210-2122) (Received 3 July 1985) An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum. 2. This carotenoprotein contains: (I) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups. 3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana. 4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein. Abstract--1.
INTRODUCTION C a r o t e n o i d - p r o t e i n complexes are described to occur widely in invertebrate phyla (Cheesman et al., 1967; Zagalsky, 1976). Two different types of c a r o t e n o i d protein complexes may be recognized: those in which variable amounts of carotenoids are associated with a lipo-(glyco-) protein and the true carotenoproteins where the carotenoids are bound stoichiometrically to a simple protein or glycoprotein (Zagalsky, 1976). The described carotenoproteins of insects are commonly associated also with other pigment types such as lutein, mesobiliverdin, anthocyanin and bile pigments (Cheesman et al., 1976). Lipophorin, the major lipoprotein of insect haemolymph, is bound to variable amounts of carotenoids (Chino and Kitasawa, 1981). To our knowledge the violet carotenoprotein of R. americana (Terra et al., 1981), is the first true haem o l y m p h carotenoprotein of insects so far described. Its mol. wt is i 57,000 and it exhibits an isoelectric pH of 5.5. The protein is a tetramer of four identical polypeptides (mol. wt 41,500) bound to three mols of echinenone and one tool of canthaxanthin (Terra et al., 1981). The carotenoprotein is one of the major protein components in the haemolymph of R. americana (Bianchi and Terra, 1976). We were able to describe a purification procedure for the carotenoprotein which permitted an immunological study of the occurrence of related proteins in other species of the Rhynchosciara genus. We also present amino acid composition data and physiological characteristics of this peculiar Rhynchosciara protein. MATERIALS AND METHODS
Animals Rhynchosciara americana was reared in the laboratory according to Lara et al. (1965). The division of the fourth 637
instar adopted here was that proposed by Terra et al. (1973). Rhynchosciara hollaenderi and the other species of this genus, not identified, were collected in banana orchards and were maintained in the laboratory until used in the experiments. Haemolymph, ovaries and fat bodies These materials were obtained as previously described (Bianchi et al., 1982). Purification of violet carotenoprotein from the haemolymph Haemolymph, obtained from larvae at the end of the feeding stage, added of 0.02% (w/v) bromophenol blue, was submitted to electrophoresis in a 1% (w/v) agarose slab (9 ×4.5 x 0.3cm). The agarose was solubilized in 50 mM NaH2PO4-NaOH buffer, pH 7.0. This same buffer was used as reservoir buffer. The electrophoretic run, under a constant current of 10mA, was interrupted when the bromophenol blue reached the end of agarose slab. The visible violet carotenoprotein spot was cut out of the slab and the protein was eluted electrophoretically from the pieces of agarose. The purified protein was stored at -20°C. Preparation of antibody and immunochemical methods About 0.2 mg of purified violet protein, was solubilized in 1.5 ml of 25 mM Tris, 192 mM glycine buffer, pH 8.3 and emulsified with 0.5 ml Freund's complete adjuvant. This emulsion was injected subcutaneously, at multiple sites, into a rabbit. After 15 and 22 days, other similar injections (about 0.1 mg of protein), but with Freund's incomplete adjuvant were administered. Ten days after the last injections the rabbit was bled and 0.01% NaN 3 was added to the serum obtained, before storage at -70°C. Double immunodiffusion was carried out according to the method of Ouchterlony (1968) using 1% noble agar in 25 mM Tris, 192 mM glycine buffer, pH 8.3, 0.1 mM phenylthiocarbamide (PTC) and 0.01% (w/v) NaN 3. Immunoelectrophoresis was made over microscope slides in 1% (w/v) agarose buffered with 50 mM NaH2PO4-NaOH, pH 7.0. The haemolymph samples were applied into 3 mm dia wells and electrophoresis was carried out with a constant current of 5 mA/slide. After the electrophoresis, antiserum (or concanavalin A solution 2 mg/ml)
638
A . G . DE BLANCH1and O. MARINOTTI
was applied into a lateral trough and the immunodiffusion was allowed to proceed overnight at room temp (25°C) in a moist chamber. Rocket immunoelectrophoresis was carried out according to Laurell (1972) using 1% (w/v) agarose in 50 mM Tris, 83 mM sodium acetate-HCl, pH 8.6 containing 4% (v/v) anti-violet protein serum. The run was carried out with 2.9 v/cm for the first hr and with 4.3 v/cm for the subsequent 17 hr. Purified violet protein was used for standardization of the method. The Rhynchosciara storage protein was quantified by rocket immunoelectrophoresis using the antiserum and the conditions previously described (Bianchi and Marinotti, 1984). The slabs of double immunodiffusion, immunoelectrophoresis and rocket immunoelectrophoresis, were washed after the runs in 0.15 M NaCI, dried and stained with 0.09% (w/v) Coomassie Blue R in ethanol acetic acid water (45: 10:45). Incorporation of labelled precursors Fat bodies (about ling) were incubated on siliconized coverslips in 20ttl of physiological medium (Terra et al., 1976) in which L-leucine was replaced by 20pCi L[3,4,5-3H(N)]leucine (142.SCi/mmol, New England Nuclear). In some experiments 32P-orthophosphate (50pCi) was added. Incorporation was conducted for 3 hr at room temp (26°C). After this time the fat bodies were removed from the medium and treated as described by Bianchi et al. (1982). The medium, in which fat bodies were incubated, was supplemented with 1/~1 of unlabelled larval haemolymph and the proteins were precipitated with trichloroacetic acid at a final conch of 10% (w/v). The precipitated proteins were collected by centrifugation at 16,000g for 10 min, at 4°C and treated in the same way as fat bodies. The treated fat bodies and proteins from the incubation medium were solubilized in sample buffer containing sodium dodecylsulphate (Bianchi et al., 1982) and used for electrophoresis and fluorography (see below). Polyacrylamide gel electrophoresis Disc electrophoresis was performed in 7% polyacrylamide gels, according to the system described by Davis (1964). Electrophoresis conditions and protein staining with amido black were as described by Bianchi et al. (1982). Slab (I0%) polyacrylamide gel electrophoresis, in the presence of SDS was carried out as described by Bianchi et al. (1982). Fluorography and autoradiography The stained polyacrylamide slabs, containing proteins labelled with [3H]leucine, were impregnated with PPO (Bonner and Laskey, 1974) and exposed at - 7 0 ° C to Sakura X-ray film. The stained polyacrylamide slabs, containing proteins labelled by [32P]orthophosphate, were dried and exposed at - 7 0 ° C to Sakura X-ray film. Amino acids determination Purified violet carotenoprotein was submitted to hydrolysis in 6 N HC1, in a sealed and evacuated ampoule, for 24 hr at 110°C. The amino acids present in the hydrolysates were analysed by the method of Spaekman et al. (1958). Quantification of violet carotenoprotein and of storage protein in the haemolymph during development For the quantification of violet carotenoproteins and of storage protein, haemolymph was collected as previously described (Bianchi et al., 1982) and diluted 150-fold with 25 mM Tris, 192 mM glycine buffer, pH 8.3, 0.05% sodium azide and 0.1 M phenylthiocarbamide. For each determination haemolymph from 10 animals was pooled. Haemolymph volumes were determined by the exsanguination method (Richardson et al., 1931), using the haemolymph density values determined by Terra et al. (1975). The violet
carotenoprotein and the storage protein were quantified by rocket immunoelectrophoresis (see above). Protein determination Protein in haemolymph was determined by the method of Lowry et al. (1951). Purified violet carotenoprotein was determined by the method of Ellman (1962). Bovine serum albumin (Sigma Chemical Co.) was used as standard.
RESULTS
Purification and further characterization o f the violet carotenoprotein W h e n R. americana larvae h a e m o l y m p h proteins were s u b m i t t e d to electrophoresis on agarose slab, at p H 7.0, the violet c a r o t e n o p r o t e i n is the protein that shows the greater m i g r a t i o n t o w a r d the a n o d e (Fig. la). Elution o f the violet c a r o t e n o p r o t e i n from the agarose slab yields a purified p r e p a r a t i o n of this protein t h a t shows only one protein fraction when analysed by p o l y a c r y l a m i d e - S D S electrophoresis (Fig. lb). The electrophoretically purified violet carotenoproteins were used to raise antibodies against it. To test the specificity of the antiserum obtained, an i m m u n o e l e c t r o p h o r e s i s was m a d e using as antigen a sample of larvae h a e m o l y m p h (Fig. lc). The result shows that only one precipitation line (violet coloured) was formed, showing t h a t the a n t i b o d y produced was specific for the violet c a r o t e n o p r o t e i n . The a m i n o acid c o m p o s i t i o n o f the violet c a r o t e n o p r o t e i n was determined (Table 1). The protein has a very low level o f histidine a n d a n o r m a l level o f tyrosine a n d phenylalanine. The analysis o f the R. americana larvae haem o l y m p h proteins by disc electrophoresis a n d staining by periodic acid a n d Schiff reagent (PAS) does Table 1. Amino acid composition of R. americana violet carotenoprotein Amino acid
~ Moles
Lys
6.55
His
traces
Arg
2,85
Asp
10.67
Thr
6.49
Ser
9.65
Glu
13.72
Pro
6.40
Gly
8,00
Ala
7,08
Cys Val
5.80
Met
1.13
lie
5,45
Leu
7.Ol
Tyr
4.14
Phe
5.04
Trp
*Not determined.
Rhynchosciara violet carotenoprotein
639
(a)
(c)
(d)
Fig. 1. (a) Electrophoresis of R. americana haemolymph proteins on agarose slab. Haemolymph from larvae at the end of feeding stage was submitted to electrophoresis in 1% (w/v) agarose slab in 50 mM NaH2PO4-NaOH buffer, pH 7.0. After the run the gel was dried and the proteins were stained with Coomassie Blue R. V, violet carotenoprotein. (b) Slab 10% polyacrylamide gel electrophoresis, in the presence of SDS, of R. americanahaemolymph and purified violet carotenoprotein. 1, R. americana second period larvae haemolymph; 2, 2.4 #g of violet carotenoprotein and 3, 4.8 #g of violet carotenoprotein. p6, protein 6 (storage protein) from R. americana; pl0, protein I0 from haemolymph; V, violet carotenoprotein. (c) Immunoelectrophoretic pattern of R. americana larval haemolymph proteins developed with anti-carotenoprotein serum (S). (1) 1 #1 and (2) 0.3 #1 of R. americana second period larval haemolymph. (d) Precipitation of R. americana haemolymph proteins by Concanavalin A. Haemolymph (1 #1) from second period larvae was submitted to electrophoresis in 1% (w/v) agarose slab buffered with 50 mM NaH2PO4-NaOH, pH 7.0. After the run, the proteins were diffused against 150 #1 of a solution 2 mg/ml of Concanavalin A (Con A). V, precipitation arc of violet carotenoprotein. not demonstrate the presence of carbohydrates bound to the violet carotenoprotein (Bianchi and Terra, 1976). However, the diffusion of the haemolymph proteins against concanavalin A produces a precipitation of the violet carotenoprotein (Fig. ld) showing therefore that this protein is a glycoprotein containing mannose and/or glucose. On the other hand, PAS staining of haemolymph proteins fractionated by electrophoresis in the presence of sodium dodecylsulphate, also shows the glycoproteic nature of the violet carotenoprotein (results not shown). The staining of the R. americana haemolymph proteins with methyl green (Cutting and Roth, 1973) shows that the violet carotenoprotein is heavily stained suggesting that it is a phosphoprotein (Fig. 2a). This fact is also evidenced by the labelling of violet carotenoprotein that is observed when larval fat bodies were incubated in the presence of [a2p]orthophosphate (Fig. 2b). The violet carotenoprotein is synthesized in vitro by the larval fat bodies and secreted to the incubation medium together with the other major Rhynehoseiara haemolymph proteins (Fig. 2c).
in the haemolymph of R. americana during the life cycle of this insect (Fig. 3b). The amount of violet carotenoprotein attains its maximal value by the middle of second period. From the end of second period until the beginning of pupal stage the amount of violet carotenoprotein is reduced from 770 to 230#g per animal. After remaining stable during the pupal life, the amount of this protein drops during the beginning of adult life. The pattern of variation of the violet carotenoprotein is similar to the pattern of variation of total haemolymph protein (Fig. 3a). During the life cycle of Rhynchosciara a great reduction of the haemolymph volume occurs (Terra et al., 1975) simultaneously with the decrease in total haemolymph protein. As a result of these variations the violet carotenoprotein concentration in haemolymph is approximately constant from the second period until the adult stage (Fig. 3c). For comparison we determined the concentration in the haemolymph of the Rhynchosciara storage protein (Bianchi and Marinotti, 1984) that is a protein utilized massively during the pupal life (Fig. 3d).
Quantification o f violet carotenoprotein during the life cycle of Rhynchosciara Violet carotenoprotein was estimated by rocket immunoelectrophoresis, (see Materials and Methods)
Occurrence of the violet carotenoprotein in other species of the Rhynchosciara genus and in R. americana fat bodies and ovaries Double immunodiffusion tests show the presence
C.B.P. 83/3B~J
A. G. DE BIANCHIand O. MARINOTTI
640 (a)
{b)
C
kp
p6 plO V
V -V ,
~!~i~ii ¸
~ii ~
I
2
I
2
I
2
Fig. 2. (a) Disc electrophoretic patterns, obtained in a 7% polyacrylamide gel, of R. americana larvae haemolymph proteins. After the electrophoretic run the cylinders were stained with Coomassie Blue R (I) or with methyl green (2). (b) Autoradiograms of fat body proteins were labelled by [32p]orthophosphate and fractionated by slab 10% polyacrylamide-SDS. After incubation in vitro with [32p]orthophosphate, the proteins of the incubated, second period larvae, fat body (1) and of the incubation medium (2) were analysed by electrophoresis and autoradiography. (c) Fluorograms of fat body proteins labelled with [3H]leucine and fractionated by slab 10% polyacrylamide SDS electrophoresis. After incubation in vitro with [3H]leucine, the proteins of incubated, second period larvae, fat body (1) and from the incubation medium (2) were analysed by electrophoresis and ftuorography. V, violet carotenoprotein; p6, protein 6 (storage protein) from R. americana; pl0, protein 10 from R. americana and Lp, major subunit from R. americana lipophorin. of the violet carotenoprotein in extracts of fat bodies obtained from larvae of second, third and fourth period and also from pharate pupa (Fig. 4). The mature ovaries of R. americana are yellow orange coloured and the possible presence in the ovaries of the violet carotenoprotein has been suggested by Bianchi and Terra (1976). However, double immunodiffusion test shows that the violet carotenoprotein is not present in the mature ovaries of R. americana (Fig. 4). The colour of R. americana ovaries may be due to carotenoids carried by the vitellin (Bianchi et al., 1982; Pereira and Bianchi, 1983) and/or by the lipophorin (yellow protein) of Rhynchosciara.
Double immunodiffusion tests with the anti-violet carotenoprotein serum show the occurrence of proteins immunologically related to R. americana violet carotenoprotein in the haemolymph of all the different Rhynchosciara species tested (Fig. 4).
DISCUSSION
The violet carotenoprotein, occurring in the haemolymph of R. americana, has an apparent mol. wt of 157,000 and is a tetramer of four identical polypeptide chains (mol. wt 41,500) to which 3 mols of
echinenone and 1 mol of canthaxanthin are bound (Terra et al., 1981). Our present results show that violet carotenoprotein is a phosphoglycoprotein containing mannose and/or glucose. The other single known example of a true carotenoprotein containing carbohydrate is ovorubin (Zagalsky, 1976). As far as we know no other true carotenoprotein containing phosphate has been described so far. The electrophoretic method of purification of violet carotenoprotein, described here, is easier to perform and much faster than the purification procedure previously described (Terra et al., 1981). The electrophoretic method also overcomes the problem of partial carotenoprotein denaturation, verified to occur in the ion exchange chromatography utilized in the previously described procedure (Terra et al., 1981). The amino acid composition of the violet carotenoprotein (Table 1) correlates well with its isoelectric pH of 5.5 (Terra et al., 1981). The violet carotenoprotein shows high contents of glutamic acid, serine, threonine and proline in agreement with the characteristic of true carotenoproteins (Zagalsky, 1976). Compared with other carotenoproteins the violet carotenoprotein shows a relative low content of aspartic acid and histidine and a higher content of leucine, isoleucine and glutamic acid.
641
Rhynchosciara violet carotenoprotein Periods
2
Periods
13 I415J6~ pupa
laduLt
2
I 3 14l~6N pupa
(c)
3
./
"\.
~
• • e.e
E
'
,,~
,o
6.
\
ZO
-/
/
ioduLt
S"~
./"
~,
\ I
I
I
I
b)
~
-
l
I
.~'",
(d)
,,.e.,~e° I
*
•
~ 5o -
\
=" 0.4
•
I
40
1 50
I 60
40
50
60
days Fig. 3. (a) R. americana total haemolymph protein per animal. Protein was determined by the method of Lowry et al. (1951). (b) Total violet carotenoprotein (v.c.p.) in R. americana haemolymph per animal. The violet carotenoprotein was determined by rocket immunoelectrophoresis. (c) Concentration of violet carotenoprotein in R. americana haemolymph. (d) Relative concentration of protein 6 (storage protein) in R. americana haemolymph. Protein 6 was determined by rocket immunoeleetrophoresis in the conditions previously described (Bianchi and Marinotti, 1984). The data are presented as percentage values in relation to the concentration attained in the haemolymph of 45 days aged animals, taken as 100%. Each point (Fig. 3a, b, c and d) is a single determination of a pooled haemolymph sample from 10 animals. The haemolymph volumes were determined by exsanguination method. The age in days after hatch is indicated as are the periods of the fourth instar, pharate pupa (PP), pupa and adult stages. Age in days
(a)
Age in
(b)
Fig. 4. Ouchterlony double diffusion tests. Centre well contained serum against violet carotenoprotein. (a) 1, R. americana haemolymph; 2, ovary soluble protein (extracted with 0.15 M NaC1, 5 mM EDTA, pH 7); 3, fat body protein from second period larvae; 4, fat body protein from third period larvae; 5, fat body protein from fifty period larvae and 6, fat body protein from pharate pupae. Fat bodies were homogenised in 25 mM Tris, 192 mM glycinebuffer, pH 8.3, 0.1mM phenylthiocarbamide and 0.01% (w/v) sodium azide. (b) 1, Rhynchosciara spl haemolymph; 2, Rhynchosciara sp2 haemolymph; 3, Rhynchosciara sp3 haemolymph; 4, R. hollaenderi haemolymph and 5, R. americana haemolymph. Haemolymph was diluted with 25 mM Tris, 192 mM glycine buffer, pH 8.3. Rhynchosciara spl, sp2 and sp3 are species not identified but deafly different from R. americana, R. hollaenderi and R. milleri.
The R. americana fat bodies, that are white in animals at the end of feeding stage, become violet coloured in animals at the stage of pharate pupa (Bianchi and Terra, 1976). The immunodiffusion tests show the presence of violet carotenoprotein in the fat bodies of pharate pupa and also in those larvae at the end of feeding stage. Since quantitative determinations were not carried out we do not know if the violet carotenoprotein is accumulated in greater amounts by the pharate pupa fat bodies. However the amount of carotenoprotein in the pharate pupa fat bodies must be not high, since we need a relatively high concentrated extract of fat bodies to see the violet carotenoprotein immunoprecipitation arc in the double immunodiffusion test. All the Rhynchosciara species analysed have in the haemolymph violet carotenoproteins that are immunologically related to the R. americana carotenoprotein. The R. milleri violet carotenoprotein is electrophoretically identical to the R. americana violet carotenoprotein (results not shown). Therefore, although we do not have immunodiffusion tests for R. milleri haemolymph we think that R. milleri violet carotenoprotein must be related to R. americana
642
A . G . DE BIANCHIand O. MARINOTTI
violet carotenoprotein. It is possible that the violet carotenoprotein is specific for the Rhynehosciara genus flies, in a similar way to that described to be the case for the R. americana protein 10 (Bianchi and Marinotti, 1985). Some Rhynchosciara species (R. papaveroi) have a light yellow coloured haemolymph and may not have a protein related to the violet carotenoprotein in the haemolymph. However the absence of dark coloured haemolymph does not apply in the absence of the apoprotein violet, since two mutants of R. americana were described in which the apoprotein violet occurs depleted of carotenoids (Terra et al., 1976). The amounts of violet carotenoprotein determined by an electrophoretic method by Bianchi and Terra (1976) are about one half of that one verified to occur by rocket immunoelectrophoresis. The violet carotenoprotein attains a value of 770 #g by larva in the second period of the development. This value is equal to that one attained by the R. americana protein 10, another major haemolymph protein that is translocated to the eggs during the end of pupal life (Marinotti and Bianchi, 1983; Bianchi and Marinotti, in press). The great consumption of haemolymph proteins verified to occur between the third period and the pupal stage of Rhynchosciara development must be due to the use of the amino acids of these proteins (including the violet protein) for the synthesis of salivary secretion proteins (Bianchi and Terra, 1976; Winter et al., 1980; Marinotti and Bianchi, 1983; Bianchi and Marinotti, 1984). It is not clear what the function is of the carotenoproteins described to occur in the invertebrates. Carotenoproteins may participate in protective colouration, photosensitivity, electron transport, enzymic activity and in the development of embryos (Cheesman et al., 1967). In the case of R. americana violet carotenoprotein, the participation of this protein in the embryo development may be ruled out since immunological tests do not demonstrate the presence of this protein in the mature ovaries. The violet carotenoprotein may have a similar function in the larvae and adults in R. americana, since its concentration is maintained during the whole life cycle of this fly. The concentration of R. americana storage protein (Fig. 3; Bianchi and Marinotti, 1984), which is a protein with no function in the adult flies, shows a very distinct pattern of variation from that one of violet carotenoprotein. In spite of the above comments the function of the violet carotenoprotein is unknown at present. Acknowledgements--This work was supported by grants from Fundaq~o de Amparo ",i Pesquisa do Estado de Silo Pauto (FAPESP), Financiadora de Estudos e Projetos (FINEP), Conv~nio No. 4.3.84.0725.00 and from CNPq No. 40/372/83. We are much indebted to Dr C. Sampaio, from the Escola Paulista de Medicina for the amino acid analysis, to M. L. C. Guimaraes for technical assistance and to Dr R. Meneghini for advice about the style of the manuscript. O. Marinotti is a graduate fellow from FAPESP and A. G. de Bianchi is a staff member of the Biochemistry Department and a research fellow from CNPq. REFERENCES
Bianchi A. G. de and Marinotti O. (1984) A storage protein
in Rhynchosciara americana (Diptera, Sciaridae). Insect Biochem. 14, 453 461. Bianchi A. G. de and Marinotti O. A specific protein in the genus Rhynehosciara (Diptera, Sciaridae). Experientia. In press. Bianchi A. G. de and Terra W. R. (1976) Haemolymph protein patterns during the spinning stage and metamorphosis of Rhynchosciara americana. J. Insect Physiol. 22, 535-540. Bianchi A. G. de, Winter C. E. and Terra W. R. (1982) Vitellogenins and other haemolymph proteins involved in the oogenesis of Rhynchosciara americana. Insect Biochem. 12, 177 184. Bonner W. M. and Laskey R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Bioehem. 46, 83 88. Cheesman D. F., Lee W. L. and Zagalsky P. F. (1967) Carotenoprotein in invertebrates. Biol. Rev. 42, 132 160. Chino H. and Kitasawa K. (1981) Diacylglycerol-carrying lipoprotein of haemolymph of the locust and some insects. J. Lipid Res. 22, 1042 1052. Cutting J. A. and Roth T. F. (1973) Staining of phosphoprotein on acrylamide gel electropherograms. Analyt. Biochem. 54, 386-394. Davis B. J. (1964) Disc electrophoresis--II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121,404 427. Ellman G. L. (1962) The Biuret reaction: changes in the u.v. absorption spectra and its application to the determination of the peptide bonds. Analyt. Biochem. 3, 40 48. Lara F. J. S., Tamaki H. and Pavan C. (1965) Laboratory culture of Rhynehosciara angelae. Am. Nat. 99, 189 191. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Marinotti O. and Bianchi A. G. de (1983) A larval haemolymph protein in the eggs of Rhynchoseiara americana. Insect Bioehem. 13, 647 653. Ouchterlony O. (1968) Handbook of Immunodiffusion and Immunoelectrophoresis. Ann. Arbor Science Publisher, Ann. Arbor. Pereira S. D. and Bianchi A. G. de (1983) Vitellogenin and vitellin of Rhynchoseiara americana: further characterization and time of synthesis. Insect Biochem. 13, 323 332. Richardson C. H., Burdette R. C. and Eagleson C. W. (1931) Blood volume larval. Ann. ent. Soc. Am. 24, 503 506. Spackman D. H., Stein W. H. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190 1206. Terra W. R., Bianchi A. G. de, Gambarini A. G. and Lara F. J. S. (1973) Haemolymph amino acids and related compounds during coccoon production by the larvae of the fly Rhynchosciara americana. J. Insect Physiol. 19, 2097 2106. Terra W. R., Bianchi A. G. de, Gambarini A. G., Pueyo M. T. and Lara F. J. S. (1976) A medium for short-term incubation of salivary glands based on haemolymph composition of the fly Rhynchosciara americana. Ci~nc. Cult., S. Pad. 28, 654-657. Terra W. R., Ferreira C. and Bianchi A. G. de (1975) Distribution of nutrient reserves during spinning in tissues of the larva of the fly Rhynchosciara americana. J. Insect Physiol. 21, 1501-1509. Terra W. R., Ferreira C., Bianchi A. G. de and Zinner K. (1981) A violet carotenoprotein, containing echinenone, isolated from the haemolymph of the fly Rhynchosciara americana. Comp. Biochem. Physiol. 68B, 89 93. Winter C. E., Bianchi A. G. de, Terra W. R. and Lara F. J. S. (1980) Protein synthesis in the salivary glands of Rhynchosciara americana. Devl Biol. 75, 1-12. Zagalsky P. F. (1976) Carotenoprotein-protein complexes. Pure appl. Chem. 47, 103-120.
0305-0491/86 $3,00+0.00 (C) 1986 Pergamon Press Ltd
PHYSIOLOGICAL PATTERN A N D F U R T H E R C H A R A C T E R I Z A T I O N OF T H E H A E M O L Y M P H V I O L E T C A R O T E N O P R O T E I N OF T H E F L Y RHYNCHOSCIARA AMERICANA A. G. DE OlANCIII and O. MARINOTTI Departamento de Bioquimica, Instituto de Quimica, Universidade de S~o Paulo, SP., C.P. 20780, CEP 01498--S~o Paulo, Brasil (Tel: 210-2122) (Received 3 July 1985) An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum. 2. This carotenoprotein contains: (I) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups. 3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana. 4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein. Abstract--1.
INTRODUCTION C a r o t e n o i d - p r o t e i n complexes are described to occur widely in invertebrate phyla (Cheesman et al., 1967; Zagalsky, 1976). Two different types of c a r o t e n o i d protein complexes may be recognized: those in which variable amounts of carotenoids are associated with a lipo-(glyco-) protein and the true carotenoproteins where the carotenoids are bound stoichiometrically to a simple protein or glycoprotein (Zagalsky, 1976). The described carotenoproteins of insects are commonly associated also with other pigment types such as lutein, mesobiliverdin, anthocyanin and bile pigments (Cheesman et al., 1976). Lipophorin, the major lipoprotein of insect haemolymph, is bound to variable amounts of carotenoids (Chino and Kitasawa, 1981). To our knowledge the violet carotenoprotein of R. americana (Terra et al., 1981), is the first true haem o l y m p h carotenoprotein of insects so far described. Its mol. wt is i 57,000 and it exhibits an isoelectric pH of 5.5. The protein is a tetramer of four identical polypeptides (mol. wt 41,500) bound to three mols of echinenone and one tool of canthaxanthin (Terra et al., 1981). The carotenoprotein is one of the major protein components in the haemolymph of R. americana (Bianchi and Terra, 1976). We were able to describe a purification procedure for the carotenoprotein which permitted an immunological study of the occurrence of related proteins in other species of the Rhynchosciara genus. We also present amino acid composition data and physiological characteristics of this peculiar Rhynchosciara protein. MATERIALS AND METHODS
Animals Rhynchosciara americana was reared in the laboratory according to Lara et al. (1965). The division of the fourth 637
instar adopted here was that proposed by Terra et al. (1973). Rhynchosciara hollaenderi and the other species of this genus, not identified, were collected in banana orchards and were maintained in the laboratory until used in the experiments. Haemolymph, ovaries and fat bodies These materials were obtained as previously described (Bianchi et al., 1982). Purification of violet carotenoprotein from the haemolymph Haemolymph, obtained from larvae at the end of the feeding stage, added of 0.02% (w/v) bromophenol blue, was submitted to electrophoresis in a 1% (w/v) agarose slab (9 ×4.5 x 0.3cm). The agarose was solubilized in 50 mM NaH2PO4-NaOH buffer, pH 7.0. This same buffer was used as reservoir buffer. The electrophoretic run, under a constant current of 10mA, was interrupted when the bromophenol blue reached the end of agarose slab. The visible violet carotenoprotein spot was cut out of the slab and the protein was eluted electrophoretically from the pieces of agarose. The purified protein was stored at -20°C. Preparation of antibody and immunochemical methods About 0.2 mg of purified violet protein, was solubilized in 1.5 ml of 25 mM Tris, 192 mM glycine buffer, pH 8.3 and emulsified with 0.5 ml Freund's complete adjuvant. This emulsion was injected subcutaneously, at multiple sites, into a rabbit. After 15 and 22 days, other similar injections (about 0.1 mg of protein), but with Freund's incomplete adjuvant were administered. Ten days after the last injections the rabbit was bled and 0.01% NaN 3 was added to the serum obtained, before storage at -70°C. Double immunodiffusion was carried out according to the method of Ouchterlony (1968) using 1% noble agar in 25 mM Tris, 192 mM glycine buffer, pH 8.3, 0.1 mM phenylthiocarbamide (PTC) and 0.01% (w/v) NaN 3. Immunoelectrophoresis was made over microscope slides in 1% (w/v) agarose buffered with 50 mM NaH2PO4-NaOH, pH 7.0. The haemolymph samples were applied into 3 mm dia wells and electrophoresis was carried out with a constant current of 5 mA/slide. After the electrophoresis, antiserum (or concanavalin A solution 2 mg/ml)
638
A . G . DE BLANCH1and O. MARINOTTI
was applied into a lateral trough and the immunodiffusion was allowed to proceed overnight at room temp (25°C) in a moist chamber. Rocket immunoelectrophoresis was carried out according to Laurell (1972) using 1% (w/v) agarose in 50 mM Tris, 83 mM sodium acetate-HCl, pH 8.6 containing 4% (v/v) anti-violet protein serum. The run was carried out with 2.9 v/cm for the first hr and with 4.3 v/cm for the subsequent 17 hr. Purified violet protein was used for standardization of the method. The Rhynchosciara storage protein was quantified by rocket immunoelectrophoresis using the antiserum and the conditions previously described (Bianchi and Marinotti, 1984). The slabs of double immunodiffusion, immunoelectrophoresis and rocket immunoelectrophoresis, were washed after the runs in 0.15 M NaCI, dried and stained with 0.09% (w/v) Coomassie Blue R in ethanol acetic acid water (45: 10:45). Incorporation of labelled precursors Fat bodies (about ling) were incubated on siliconized coverslips in 20ttl of physiological medium (Terra et al., 1976) in which L-leucine was replaced by 20pCi L[3,4,5-3H(N)]leucine (142.SCi/mmol, New England Nuclear). In some experiments 32P-orthophosphate (50pCi) was added. Incorporation was conducted for 3 hr at room temp (26°C). After this time the fat bodies were removed from the medium and treated as described by Bianchi et al. (1982). The medium, in which fat bodies were incubated, was supplemented with 1/~1 of unlabelled larval haemolymph and the proteins were precipitated with trichloroacetic acid at a final conch of 10% (w/v). The precipitated proteins were collected by centrifugation at 16,000g for 10 min, at 4°C and treated in the same way as fat bodies. The treated fat bodies and proteins from the incubation medium were solubilized in sample buffer containing sodium dodecylsulphate (Bianchi et al., 1982) and used for electrophoresis and fluorography (see below). Polyacrylamide gel electrophoresis Disc electrophoresis was performed in 7% polyacrylamide gels, according to the system described by Davis (1964). Electrophoresis conditions and protein staining with amido black were as described by Bianchi et al. (1982). Slab (I0%) polyacrylamide gel electrophoresis, in the presence of SDS was carried out as described by Bianchi et al. (1982). Fluorography and autoradiography The stained polyacrylamide slabs, containing proteins labelled with [3H]leucine, were impregnated with PPO (Bonner and Laskey, 1974) and exposed at - 7 0 ° C to Sakura X-ray film. The stained polyacrylamide slabs, containing proteins labelled by [32P]orthophosphate, were dried and exposed at - 7 0 ° C to Sakura X-ray film. Amino acids determination Purified violet carotenoprotein was submitted to hydrolysis in 6 N HC1, in a sealed and evacuated ampoule, for 24 hr at 110°C. The amino acids present in the hydrolysates were analysed by the method of Spaekman et al. (1958). Quantification of violet carotenoprotein and of storage protein in the haemolymph during development For the quantification of violet carotenoproteins and of storage protein, haemolymph was collected as previously described (Bianchi et al., 1982) and diluted 150-fold with 25 mM Tris, 192 mM glycine buffer, pH 8.3, 0.05% sodium azide and 0.1 M phenylthiocarbamide. For each determination haemolymph from 10 animals was pooled. Haemolymph volumes were determined by the exsanguination method (Richardson et al., 1931), using the haemolymph density values determined by Terra et al. (1975). The violet
carotenoprotein and the storage protein were quantified by rocket immunoelectrophoresis (see above). Protein determination Protein in haemolymph was determined by the method of Lowry et al. (1951). Purified violet carotenoprotein was determined by the method of Ellman (1962). Bovine serum albumin (Sigma Chemical Co.) was used as standard.
RESULTS
Purification and further characterization o f the violet carotenoprotein W h e n R. americana larvae h a e m o l y m p h proteins were s u b m i t t e d to electrophoresis on agarose slab, at p H 7.0, the violet c a r o t e n o p r o t e i n is the protein that shows the greater m i g r a t i o n t o w a r d the a n o d e (Fig. la). Elution o f the violet c a r o t e n o p r o t e i n from the agarose slab yields a purified p r e p a r a t i o n of this protein t h a t shows only one protein fraction when analysed by p o l y a c r y l a m i d e - S D S electrophoresis (Fig. lb). The electrophoretically purified violet carotenoproteins were used to raise antibodies against it. To test the specificity of the antiserum obtained, an i m m u n o e l e c t r o p h o r e s i s was m a d e using as antigen a sample of larvae h a e m o l y m p h (Fig. lc). The result shows that only one precipitation line (violet coloured) was formed, showing t h a t the a n t i b o d y produced was specific for the violet c a r o t e n o p r o t e i n . The a m i n o acid c o m p o s i t i o n o f the violet c a r o t e n o p r o t e i n was determined (Table 1). The protein has a very low level o f histidine a n d a n o r m a l level o f tyrosine a n d phenylalanine. The analysis o f the R. americana larvae haem o l y m p h proteins by disc electrophoresis a n d staining by periodic acid a n d Schiff reagent (PAS) does Table 1. Amino acid composition of R. americana violet carotenoprotein Amino acid
~ Moles
Lys
6.55
His
traces
Arg
2,85
Asp
10.67
Thr
6.49
Ser
9.65
Glu
13.72
Pro
6.40
Gly
8,00
Ala
7,08
Cys Val
5.80
Met
1.13
lie
5,45
Leu
7.Ol
Tyr
4.14
Phe
5.04
Trp
*Not determined.
Rhynchosciara violet carotenoprotein
639
(a)
(c)
(d)
Fig. 1. (a) Electrophoresis of R. americana haemolymph proteins on agarose slab. Haemolymph from larvae at the end of feeding stage was submitted to electrophoresis in 1% (w/v) agarose slab in 50 mM NaH2PO4-NaOH buffer, pH 7.0. After the run the gel was dried and the proteins were stained with Coomassie Blue R. V, violet carotenoprotein. (b) Slab 10% polyacrylamide gel electrophoresis, in the presence of SDS, of R. americanahaemolymph and purified violet carotenoprotein. 1, R. americana second period larvae haemolymph; 2, 2.4 #g of violet carotenoprotein and 3, 4.8 #g of violet carotenoprotein. p6, protein 6 (storage protein) from R. americana; pl0, protein I0 from haemolymph; V, violet carotenoprotein. (c) Immunoelectrophoretic pattern of R. americana larval haemolymph proteins developed with anti-carotenoprotein serum (S). (1) 1 #1 and (2) 0.3 #1 of R. americana second period larval haemolymph. (d) Precipitation of R. americana haemolymph proteins by Concanavalin A. Haemolymph (1 #1) from second period larvae was submitted to electrophoresis in 1% (w/v) agarose slab buffered with 50 mM NaH2PO4-NaOH, pH 7.0. After the run, the proteins were diffused against 150 #1 of a solution 2 mg/ml of Concanavalin A (Con A). V, precipitation arc of violet carotenoprotein. not demonstrate the presence of carbohydrates bound to the violet carotenoprotein (Bianchi and Terra, 1976). However, the diffusion of the haemolymph proteins against concanavalin A produces a precipitation of the violet carotenoprotein (Fig. ld) showing therefore that this protein is a glycoprotein containing mannose and/or glucose. On the other hand, PAS staining of haemolymph proteins fractionated by electrophoresis in the presence of sodium dodecylsulphate, also shows the glycoproteic nature of the violet carotenoprotein (results not shown). The staining of the R. americana haemolymph proteins with methyl green (Cutting and Roth, 1973) shows that the violet carotenoprotein is heavily stained suggesting that it is a phosphoprotein (Fig. 2a). This fact is also evidenced by the labelling of violet carotenoprotein that is observed when larval fat bodies were incubated in the presence of [a2p]orthophosphate (Fig. 2b). The violet carotenoprotein is synthesized in vitro by the larval fat bodies and secreted to the incubation medium together with the other major Rhynehoseiara haemolymph proteins (Fig. 2c).
in the haemolymph of R. americana during the life cycle of this insect (Fig. 3b). The amount of violet carotenoprotein attains its maximal value by the middle of second period. From the end of second period until the beginning of pupal stage the amount of violet carotenoprotein is reduced from 770 to 230#g per animal. After remaining stable during the pupal life, the amount of this protein drops during the beginning of adult life. The pattern of variation of the violet carotenoprotein is similar to the pattern of variation of total haemolymph protein (Fig. 3a). During the life cycle of Rhynchosciara a great reduction of the haemolymph volume occurs (Terra et al., 1975) simultaneously with the decrease in total haemolymph protein. As a result of these variations the violet carotenoprotein concentration in haemolymph is approximately constant from the second period until the adult stage (Fig. 3c). For comparison we determined the concentration in the haemolymph of the Rhynchosciara storage protein (Bianchi and Marinotti, 1984) that is a protein utilized massively during the pupal life (Fig. 3d).
Quantification o f violet carotenoprotein during the life cycle of Rhynchosciara Violet carotenoprotein was estimated by rocket immunoelectrophoresis, (see Materials and Methods)
Occurrence of the violet carotenoprotein in other species of the Rhynchosciara genus and in R. americana fat bodies and ovaries Double immunodiffusion tests show the presence
C.B.P. 83/3B~J
A. G. DE BIANCHIand O. MARINOTTI
640 (a)
{b)
C
kp
p6 plO V
V -V ,
~!~i~ii ¸
~ii ~
I
2
I
2
I
2
Fig. 2. (a) Disc electrophoretic patterns, obtained in a 7% polyacrylamide gel, of R. americana larvae haemolymph proteins. After the electrophoretic run the cylinders were stained with Coomassie Blue R (I) or with methyl green (2). (b) Autoradiograms of fat body proteins were labelled by [32p]orthophosphate and fractionated by slab 10% polyacrylamide-SDS. After incubation in vitro with [32p]orthophosphate, the proteins of the incubated, second period larvae, fat body (1) and of the incubation medium (2) were analysed by electrophoresis and autoradiography. (c) Fluorograms of fat body proteins labelled with [3H]leucine and fractionated by slab 10% polyacrylamide SDS electrophoresis. After incubation in vitro with [3H]leucine, the proteins of incubated, second period larvae, fat body (1) and from the incubation medium (2) were analysed by electrophoresis and ftuorography. V, violet carotenoprotein; p6, protein 6 (storage protein) from R. americana; pl0, protein 10 from R. americana and Lp, major subunit from R. americana lipophorin. of the violet carotenoprotein in extracts of fat bodies obtained from larvae of second, third and fourth period and also from pharate pupa (Fig. 4). The mature ovaries of R. americana are yellow orange coloured and the possible presence in the ovaries of the violet carotenoprotein has been suggested by Bianchi and Terra (1976). However, double immunodiffusion test shows that the violet carotenoprotein is not present in the mature ovaries of R. americana (Fig. 4). The colour of R. americana ovaries may be due to carotenoids carried by the vitellin (Bianchi et al., 1982; Pereira and Bianchi, 1983) and/or by the lipophorin (yellow protein) of Rhynchosciara.
Double immunodiffusion tests with the anti-violet carotenoprotein serum show the occurrence of proteins immunologically related to R. americana violet carotenoprotein in the haemolymph of all the different Rhynchosciara species tested (Fig. 4).
DISCUSSION
The violet carotenoprotein, occurring in the haemolymph of R. americana, has an apparent mol. wt of 157,000 and is a tetramer of four identical polypeptide chains (mol. wt 41,500) to which 3 mols of
echinenone and 1 mol of canthaxanthin are bound (Terra et al., 1981). Our present results show that violet carotenoprotein is a phosphoglycoprotein containing mannose and/or glucose. The other single known example of a true carotenoprotein containing carbohydrate is ovorubin (Zagalsky, 1976). As far as we know no other true carotenoprotein containing phosphate has been described so far. The electrophoretic method of purification of violet carotenoprotein, described here, is easier to perform and much faster than the purification procedure previously described (Terra et al., 1981). The electrophoretic method also overcomes the problem of partial carotenoprotein denaturation, verified to occur in the ion exchange chromatography utilized in the previously described procedure (Terra et al., 1981). The amino acid composition of the violet carotenoprotein (Table 1) correlates well with its isoelectric pH of 5.5 (Terra et al., 1981). The violet carotenoprotein shows high contents of glutamic acid, serine, threonine and proline in agreement with the characteristic of true carotenoproteins (Zagalsky, 1976). Compared with other carotenoproteins the violet carotenoprotein shows a relative low content of aspartic acid and histidine and a higher content of leucine, isoleucine and glutamic acid.
641
Rhynchosciara violet carotenoprotein Periods
2
Periods
13 I415J6~ pupa
laduLt
2
I 3 14l~6N pupa
(c)
3
./
"\.
~
• • e.e
E
'
,,~
,o
6.
\
ZO
-/
/
ioduLt
S"~
./"
~,
\ I
I
I
I
b)
~
-
l
I
.~'",
(d)
,,.e.,~e° I
*
•
~ 5o -
\
=" 0.4
•
I
40
1 50
I 60
40
50
60
days Fig. 3. (a) R. americana total haemolymph protein per animal. Protein was determined by the method of Lowry et al. (1951). (b) Total violet carotenoprotein (v.c.p.) in R. americana haemolymph per animal. The violet carotenoprotein was determined by rocket immunoelectrophoresis. (c) Concentration of violet carotenoprotein in R. americana haemolymph. (d) Relative concentration of protein 6 (storage protein) in R. americana haemolymph. Protein 6 was determined by rocket immunoeleetrophoresis in the conditions previously described (Bianchi and Marinotti, 1984). The data are presented as percentage values in relation to the concentration attained in the haemolymph of 45 days aged animals, taken as 100%. Each point (Fig. 3a, b, c and d) is a single determination of a pooled haemolymph sample from 10 animals. The haemolymph volumes were determined by exsanguination method. The age in days after hatch is indicated as are the periods of the fourth instar, pharate pupa (PP), pupa and adult stages. Age in days
(a)
Age in
(b)
Fig. 4. Ouchterlony double diffusion tests. Centre well contained serum against violet carotenoprotein. (a) 1, R. americana haemolymph; 2, ovary soluble protein (extracted with 0.15 M NaC1, 5 mM EDTA, pH 7); 3, fat body protein from second period larvae; 4, fat body protein from third period larvae; 5, fat body protein from fifty period larvae and 6, fat body protein from pharate pupae. Fat bodies were homogenised in 25 mM Tris, 192 mM glycinebuffer, pH 8.3, 0.1mM phenylthiocarbamide and 0.01% (w/v) sodium azide. (b) 1, Rhynchosciara spl haemolymph; 2, Rhynchosciara sp2 haemolymph; 3, Rhynchosciara sp3 haemolymph; 4, R. hollaenderi haemolymph and 5, R. americana haemolymph. Haemolymph was diluted with 25 mM Tris, 192 mM glycine buffer, pH 8.3. Rhynchosciara spl, sp2 and sp3 are species not identified but deafly different from R. americana, R. hollaenderi and R. milleri.
The R. americana fat bodies, that are white in animals at the end of feeding stage, become violet coloured in animals at the stage of pharate pupa (Bianchi and Terra, 1976). The immunodiffusion tests show the presence of violet carotenoprotein in the fat bodies of pharate pupa and also in those larvae at the end of feeding stage. Since quantitative determinations were not carried out we do not know if the violet carotenoprotein is accumulated in greater amounts by the pharate pupa fat bodies. However the amount of carotenoprotein in the pharate pupa fat bodies must be not high, since we need a relatively high concentrated extract of fat bodies to see the violet carotenoprotein immunoprecipitation arc in the double immunodiffusion test. All the Rhynchosciara species analysed have in the haemolymph violet carotenoproteins that are immunologically related to the R. americana carotenoprotein. The R. milleri violet carotenoprotein is electrophoretically identical to the R. americana violet carotenoprotein (results not shown). Therefore, although we do not have immunodiffusion tests for R. milleri haemolymph we think that R. milleri violet carotenoprotein must be related to R. americana
642
A . G . DE BIANCHIand O. MARINOTTI
violet carotenoprotein. It is possible that the violet carotenoprotein is specific for the Rhynehosciara genus flies, in a similar way to that described to be the case for the R. americana protein 10 (Bianchi and Marinotti, 1985). Some Rhynchosciara species (R. papaveroi) have a light yellow coloured haemolymph and may not have a protein related to the violet carotenoprotein in the haemolymph. However the absence of dark coloured haemolymph does not apply in the absence of the apoprotein violet, since two mutants of R. americana were described in which the apoprotein violet occurs depleted of carotenoids (Terra et al., 1976). The amounts of violet carotenoprotein determined by an electrophoretic method by Bianchi and Terra (1976) are about one half of that one verified to occur by rocket immunoelectrophoresis. The violet carotenoprotein attains a value of 770 #g by larva in the second period of the development. This value is equal to that one attained by the R. americana protein 10, another major haemolymph protein that is translocated to the eggs during the end of pupal life (Marinotti and Bianchi, 1983; Bianchi and Marinotti, in press). The great consumption of haemolymph proteins verified to occur between the third period and the pupal stage of Rhynchosciara development must be due to the use of the amino acids of these proteins (including the violet protein) for the synthesis of salivary secretion proteins (Bianchi and Terra, 1976; Winter et al., 1980; Marinotti and Bianchi, 1983; Bianchi and Marinotti, 1984). It is not clear what the function is of the carotenoproteins described to occur in the invertebrates. Carotenoproteins may participate in protective colouration, photosensitivity, electron transport, enzymic activity and in the development of embryos (Cheesman et al., 1967). In the case of R. americana violet carotenoprotein, the participation of this protein in the embryo development may be ruled out since immunological tests do not demonstrate the presence of this protein in the mature ovaries. The violet carotenoprotein may have a similar function in the larvae and adults in R. americana, since its concentration is maintained during the whole life cycle of this fly. The concentration of R. americana storage protein (Fig. 3; Bianchi and Marinotti, 1984), which is a protein with no function in the adult flies, shows a very distinct pattern of variation from that one of violet carotenoprotein. In spite of the above comments the function of the violet carotenoprotein is unknown at present. Acknowledgements--This work was supported by grants from Fundaq~o de Amparo ",i Pesquisa do Estado de Silo Pauto (FAPESP), Financiadora de Estudos e Projetos (FINEP), Conv~nio No. 4.3.84.0725.00 and from CNPq No. 40/372/83. We are much indebted to Dr C. Sampaio, from the Escola Paulista de Medicina for the amino acid analysis, to M. L. C. Guimaraes for technical assistance and to Dr R. Meneghini for advice about the style of the manuscript. O. Marinotti is a graduate fellow from FAPESP and A. G. de Bianchi is a staff member of the Biochemistry Department and a research fellow from CNPq. REFERENCES
Bianchi A. G. de and Marinotti O. (1984) A storage protein
in Rhynchosciara americana (Diptera, Sciaridae). Insect Biochem. 14, 453 461. Bianchi A. G. de and Marinotti O. A specific protein in the genus Rhynehosciara (Diptera, Sciaridae). Experientia. In press. Bianchi A. G. de and Terra W. R. (1976) Haemolymph protein patterns during the spinning stage and metamorphosis of Rhynchosciara americana. J. Insect Physiol. 22, 535-540. Bianchi A. G. de, Winter C. E. and Terra W. R. (1982) Vitellogenins and other haemolymph proteins involved in the oogenesis of Rhynchosciara americana. Insect Biochem. 12, 177 184. Bonner W. M. and Laskey R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Bioehem. 46, 83 88. Cheesman D. F., Lee W. L. and Zagalsky P. F. (1967) Carotenoprotein in invertebrates. Biol. Rev. 42, 132 160. Chino H. and Kitasawa K. (1981) Diacylglycerol-carrying lipoprotein of haemolymph of the locust and some insects. J. Lipid Res. 22, 1042 1052. Cutting J. A. and Roth T. F. (1973) Staining of phosphoprotein on acrylamide gel electropherograms. Analyt. Biochem. 54, 386-394. Davis B. J. (1964) Disc electrophoresis--II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121,404 427. Ellman G. L. (1962) The Biuret reaction: changes in the u.v. absorption spectra and its application to the determination of the peptide bonds. Analyt. Biochem. 3, 40 48. Lara F. J. S., Tamaki H. and Pavan C. (1965) Laboratory culture of Rhynehosciara angelae. Am. Nat. 99, 189 191. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Marinotti O. and Bianchi A. G. de (1983) A larval haemolymph protein in the eggs of Rhynchoseiara americana. Insect Bioehem. 13, 647 653. Ouchterlony O. (1968) Handbook of Immunodiffusion and Immunoelectrophoresis. Ann. Arbor Science Publisher, Ann. Arbor. Pereira S. D. and Bianchi A. G. de (1983) Vitellogenin and vitellin of Rhynchoseiara americana: further characterization and time of synthesis. Insect Biochem. 13, 323 332. Richardson C. H., Burdette R. C. and Eagleson C. W. (1931) Blood volume larval. Ann. ent. Soc. Am. 24, 503 506. Spackman D. H., Stein W. H. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190 1206. Terra W. R., Bianchi A. G. de, Gambarini A. G. and Lara F. J. S. (1973) Haemolymph amino acids and related compounds during coccoon production by the larvae of the fly Rhynchosciara americana. J. Insect Physiol. 19, 2097 2106. Terra W. R., Bianchi A. G. de, Gambarini A. G., Pueyo M. T. and Lara F. J. S. (1976) A medium for short-term incubation of salivary glands based on haemolymph composition of the fly Rhynchosciara americana. Ci~nc. Cult., S. Pad. 28, 654-657. Terra W. R., Ferreira C. and Bianchi A. G. de (1975) Distribution of nutrient reserves during spinning in tissues of the larva of the fly Rhynchosciara americana. J. Insect Physiol. 21, 1501-1509. Terra W. R., Ferreira C., Bianchi A. G. de and Zinner K. (1981) A violet carotenoprotein, containing echinenone, isolated from the haemolymph of the fly Rhynchosciara americana. Comp. Biochem. Physiol. 68B, 89 93. Winter C. E., Bianchi A. G. de, Terra W. R. and Lara F. J. S. (1980) Protein synthesis in the salivary glands of Rhynchosciara americana. Devl Biol. 75, 1-12. Zagalsky P. F. (1976) Carotenoprotein-protein complexes. Pure appl. Chem. 47, 103-120.