Carotenoids from midgut and from haemolymph proteins of Rhynchosciara (Diptera: sciaridae) and their metabolic implications

Comp. Biochem. Physiol., Vol. 66B, pp. 491 to 497

0305-0491/80/0801-0491502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

CAROTENOIDS FROM MIDGUT AND FROM HAEMOLYMPH PROTEINS OF RHYNCHOSCIARA (DIPTERA: SCIARIDAE) AND THEIR METABOLIC IMPLICATIONS W. R. TERRA, C. FERREIRAand A. G. DE BIANCHI Departamento de Bioquimica, Instituto de Quimica, Universidade de S~o Paulo, C.P. 20780, S~to Paulo, Brasil (Received 29 November 1979) Abslract--1. The isolation, identification and quantitation of the chief haemolymph and midgut carotenoids of Rhynchosciara americana larvae was accomplished. 2. Major haemolymph carotenoids were: echinenone, canthaxanthin, cryptoxanthin and fl-carotene; minor carotenoids being isocryptoxanthin and 4-hydroxy-4'-keto fl-carotene. 3. Midgut stores large amounts of several carotenoids from which absorbed dietary fl-carotene is the major one. 4. The haemolymph yellow lipoprotein is a protein (molecular weight 287,000) composed of two subunits (molecular weights 176,000 and 101,000)associated with lipids, where the following carotenoids are solubilized: echinenone, fl-carotene and cryptoxanthin. 5. Tile data are discussed in the light of known color mutants of R. americana and it is proposed that haemolymph carotenoids are derived from dietary fl-carotene absorbed and oxidized in the midgut, and that they are loaded onto haemolymph proteins directly from midgut.

INTRODUCTION

The structure and occurrence of carotenoids among living beings are well known (review in Weedon, 1971). Most efforts in this field are, in the present, directed to two main lines of research: the metabolism of carotenoids and their function. Metabolism is studied chiefly by the use of labelled precursors and/or metabolic mutants, and function is sometimes approached by attempts to understand the structure of carotenoid-protein complexes, both in plants and in animals (Thommen, 1971; Krinsky, 1971; Goodwin, 1971). The color of the larvae of Rhynchosciara americana is determined by their haemolymph carotenoids (Terra et al., 1974). Disorders in the carotenoid metabolism in R. americana larvae were proposed to explain some color mutants found, basing in a preliminary identification of their haemolymph carotenoids (Terra et al., 1976). In this paper the quantitation and identification of the chief carotenoids of the R. americana haemolymph and midgut were accomplished by using several criteria, and a procedure for the isolation of carotenoidlinked proteins was developed. The results confirmed the general conclusions of our previous paper (Terra et al., 1976) and supported the assertion the oxidation of dietary carotenoids takes place in the midgut, from which they are loaded onto haemolymph chromoproteins. MATERIALS AND METHODS

Materials

/Lcarotene, echinenone and canthaxanthin were a gift from the Brazilian branch of the Hoffman-La Roche and ¢.B.P. 66/4~

D

491

Co, Basle, Switzerland. Isozeaxanthin and 4-hydroxy-4'keto-fl-carotene were obtained by respectively total and partial sodium borohydride reduction of canthaxanthin, whereas isocryptoxanthin came from echinenone by reduction. Silica gel G (Stahl) and Aluminium oxide G (Stahl) were purchased from E. Merck, Darmstadt (Germany). Ion exchange resins came from Pharmacia Fine Chemicals (Sweden), acrylamide and bis-acrylamide from Bio-Rad Laboratories (U.S.A.) and molecular weight markers from Sigma Chemical Company (U.S.A.). All the other reagents were analytical grade reagents from E. Merck (Darmstadt, Germany) and J. T. Baker (U.S.A.). The solutions were prepared in glass double distilled water. Animals Rhynchosciara americana (Diptera, Sciaridae) were raised according to Lara et al. (1965). The division of the fourth instar adopted here was that proposed by Terra et al. (1973). Rhynchosciara milleri and Rhynchosciara sp (probably baschanti) larvae were collected in banana orchards near the coast and were maintained in the laboratory, up till they were used in the experiments, in the same way as R. americana. Polyacrylamide gel electrophoresis Electrophoresis was performed in 7% acrylamide gels according to the system described by Davis (1964). The proteins in the gels were stained with amido black (Panyims & Chalkley, 1969), and scanned in a u.v. Scanner (Joyce Lobl) as described by Davis et al. (1974). Molecular weioht of the native yellow chromoprotein The molecular weight of the yellow chromoprotein was determined in unfractionated haemolymph by measuring its electrophoretical migration in acrylamide gels of different concentrations (4.5, 5.5, 6.5 and 7.5%) (Davis, 1964) and comparing its behavior with those of proteins of known molecular weight (Hedriek & Smith, 1968).

492

W. R, TERRA, C. FERREmA and A. G. DE BIANCHI

SDS-polyacrylamide gel electrophoresis Purified yellow chromoprotein, as well as the protein used as molecular weight markers, were dissolved in sample buffer (60 mM Tris-HCl buffer, pH 6.8; 2.5~ SDS; 0.36 M fl-mercaptoethanol; 0.5 mM EDTA; 10~ glycerol and 0.005~ bromophenol blue) and heated for 1 hr at 50°C. The samples were run in a thin layer of 10~ acrylamide gel containing 0.1~ SDS, using the buffer system of Laemmli (1970) in an apparatus similar to the one described by Studier (1973). Polyacrylamide plates were stained with 0.09~o Coomassie Blue R.

Ammonium sulfate fractionation of haemolymph proteins One volume of saturated ammonium sulfate in water was slowly added with continuous stirring to one volume of haemolymph obtained as described elsewhere (Terra et al., 1974) and maintained in a water-ice bath. After 5 min, the suspension was centrifuged at 10,000g, 10rain, the supernatant was made 60% saturated in ammonium sulfate and centrifuged as before. The same procedure was repeated to sequentially obtained supernatants 70 and 80~ saturated in ammonium sulfate. The proteins precipitating in the range of 0-50, 50-60, 60-70 and 70-80~ ammonium sulfate saturation were solubilized in 2 ml Tris-glycine buffer (2.5 mM Tris, 38 mM glycine, pH 8.3), concentrated to 0.5 ml with the aid of a membrane filter (Diaflo UM-10 Amicon, Corp.) and then diafiltrated once more with 2 ml of Tris-glycine buffer. The concentrated samples were used in the determination of proteins, according to Lowry et al. (1951), in gel electrophoresis or in column chromatography.

Ion-exchange chromatography of haemolymph chromoproteins A sample of haemolymph was made 60~ saturated in ammonium sulfate and cleared by centrifugation. To the supernatant, solid ammonium sulfate was added to become 80~o saturated, and the resulting suspension was passed through a filter paper. The retained sediment was solubilized from the filter paper by the addition of 0.05 M phosphate buffer pH 6.0 and the filtrate, after being concentrated with the aid of a membrane filter (Diaflo UM-10), was loaded at the top of a DEAE-Sephadex 0.6 x 6 cm column equilibrated with 0.05 M phosphate buffer pH 6.0. Elution of the yellow chromoprotein was accomplished with 3.0 ml of 0.05 M phosphate buffer pH 6.0. In these conditions the violet chromoprotein remain attached to the column. The yellow eluate was then applied at the top of a CMC-Sephadex 0.6 x 6.0cm column equilibrated with 0.05 M phosphate buffer pH 6.0. The yellow chromoprotein was retained in the column and after passing through the column 4 ml 0.05 M phosphate buffer pH 6.0 it was eluted in 10ml 0.05M phosphate buffer pH 6.0, 0.1M NaC1.

trations of acetone in hexane. The spots were scraped off from the plates immediately after development and eluted with acetone. The acetone extracts were used to estimate the relative amounts of the different carotenoids assuming all of them have the same molar absorption at 450 nm in acetone. In preparative runs, identical acetone extracts were combined before use in the several identification steps.

RESULTS R. americana haemolymph carotenoids Figure 1 displays a thin-layer c h r o m a t o g r a m of R.

americana h a e m o l y m p h carotenoids. A similar result was obtained if the carotenoid sample was saponified before spotting in the plate, which indicated the absence of carotenoid esters. Since material in spots No. 5 and 6 was poorly resolved in silica gel thinlayers, it was scraped off from them and resolved in aluminium oxide G thin-layers (solvent: 25~o acetone in hexane) before being used in the identification steps. Table 1 shows the relative a b u n d a n c e of the different h a e m o l y m p h carotenoids. Spot No. 1 was identified as all-trans-fl-carotene, based on its absorption spectrum in hexane (absorption max: 446 nm) a n d on its migration in thin-layers of silica gel G (solvents: 7~o acetone in hexane and 25~o acetone in hexane) and aluminium oxide G (solvent: 2 5 ~ acetone in hexane) which were identical to a n authentic sample of fl-carotene. Spot No. 2 was considered to be cis-4-keto-flcarotene based on its absorption spectrum in hexane which is similar to that of echinenone, with two remarkable differences: it displays a cis-peak (Zech-

1

~m~

~

~

~

0

0

o

~

0,

0

0

.::7

Extraction, saponification, reduction and partition coefficients of carotenoids Carotenoids were extracted with chloroform-methanol (2:1, v/v), filtered through filter paper, evaporated at reduced pressure, and dissolved in the appropriate solvent before use. Saponification and reduction of carotenoids with sodium borohydride were accomplished according to Harashima (1970). The determination of partition coefficients of carotenoids between hexane and 95~ methanol were performed as described by Petracek & Zechmeister (1956). Elimination of allylic hydroxyl groups were carried out with the acid chloroform reagent according to Wallcave & Zechmeister (1953).

Thin-layer chromatography of carotenoids Carotenoid chromatograms were developed in thinlayers (250 nm thick) of silica gel G, or aluminium oxide G activated for 1 hr at l l0°C, employing different concen-

e

o

7 8

0 0

.....

X

l(

X

H

M

Y

X

H,sp

x

H,m

Fig. 1. Thin-layer chromatography of the catotenoids from the haemolymph (H), midgut (M) and yellow lipoprotein (Y) of R. americana larvae and from the haemolymph of Rhynchosciara sp (Hsp) and R. milleri (Hm). The samples were run in silica gel G plates employing 7 ~ acetone in hexane. The shading of the spots is proportional to the amount of color.

493

Carotenoids from Rhynchosciara Table 1. Thin-layer chromatography on Silica gel G of carotenoids extracted from three species of Rhynchosciara Spot No. 1 2 3 4 5 6 7 8

Relative abundance (~o) R. americana Rhynchosciara sp R. milleri Haemolymph Midgut Yellowlipoprotein Haemolymph Haemolymph Carotenoid 6.2 2.5 72.6 Trace 7.9 \ 8.4 f 1.0 1.4

60.2 0 30.7 0 9.1 0 0

13.3 10.7 64.0 0 12.0 0 0 0

0.5 9.5 35.5 0.3 2.3 47.6 4.4 0

12.0 17.6 50.6 1.6 8.4 9.6 Trace 0

fl-carotene cis-4-keto E-carotene Echinenone Isocryptoxanthin Cryptoxanthin Canthaxanthin 4-hydroxy-4'keto-fl-carotene Unknown

Solvent: 7~o acetone in hexane. Spots are numbered as in Fig. 1. Data are representative values. Results vary somewhat among different preparations mainly as consequence of changes in amount of cis-4-keto fl-carotene. meister & Polgfir, 1943) at 346nm and the main visible absorption band is hypsochromically displaced (absorption max: 453 nm). Furthermore its partitition coefficient between hexane and 95~o methanol (97: 3) is similar to that one of echinenone and it occurs in variable amounts which seems to depend on the mass of echinenone and light exposure of the samples. This carotenoid seems to be an artefact and its mass should be added to that one of echinenone. Spot No. 3 seems to correspond to echinenone (4-keto-fl-carotene) considering the following: (a) its absorption spectrum has only one peak which changes to three peaks on reduction, suggesting the existence of a keto group in the position 4 (cf. Vetter et al., 1971) (b) its partition coefficient between hexane and 95~ methanol in the non-reduced (96:4) and in the reduced (87:13) form suggests it has only one ketone group before reduction, changing to one hydroxyl group after reduction (cf. Petracek & Zechmeister, 1956) (c) the absorption maxima in hexane before (459 nm) and after (448 nm) reduction agree with that one of echinenone and isocryptoxanthin (4-hydroxy-fl-carotene) (cf. Foppen, 1971), respectively (d) the reduced carotenoid changes color and migration in thin-layer chromatography after treatment with acid chloroform reagent indicating it has an allylic hydroxyl group (Wallcave & Zechmeister, 1953) (e) finally, it migrates together with an authentic sample of echinenone in thin-layers of silica gel G (solvents: 7?/o acetone in hexane; 25yo acetone in hexane) and aluminium oxide G (solvent: 25~o acetone in hexane) and after reduction it migrates together with authentic isocryptoxanthin in thin-layers of silica gel G (solvent: 25?/o acetone in hexane) and aluminium oxide G (solvent: 25~o acetone in hexane). Spot No. 5 was supposed to be cryptoxanthin (3-hydroxy-fl-carotene) since its absorption spectrum in hexane (maxima: 425, 450, 478), partition coefficient between hexane and 95~o methanol (86:14), and migration on silica gel G (solvent: 7~o acetone in hexane), before and after treatment with sodium borohydride (followed or not by exposure to the acid chloroform reagent), they do not change in accordance to the properties of cryptoxanthin (cf. Foppen, 1971). Furthermore, spot No. 5 migrates slower than isocryptoxanthin in thin-layers of silica gel G and aluminium oxide G (solvent: 7~o acetone in hexane),

in agreement with the fact that non-allylic hydroxyl are usually more polar than allylic hydroxyl groups (Krinsky, 1963). Otherwise spot No. 5 could not be the rare 2-hydroxy-fl-carotene, which is less polar than isocryptoxanthin, as judged by thin-layer chromatography in kieselguhr paper and has a partition coefficient between petroleum ether and 95~o methanol equal to 100:0 (Kjosen et al., 1972). Spot No. 6 was identified as canthaxanthin based in the following. The absorption spectrum of the material in spot No. 6 has one peak in hexane (max 465 nm) changing to three peaks (max 425, 449, 478) after borohydride reduction, while its partition coefficient between hexane and 95~ methanol changes from 61:39 to 9:91. After reduction, it was treated with the acid chloroform reagent resulting in a change in polarity and in color. These properties agree with those of authentic canthaxanthin. Furthermore the material in spot No. 6 before and after reduction migrates together with respectively authentic nonreduced and reduced canthaxanthin in thin-layers of silica gel G (solvent: 7~o acetone in hexane and 25~ acetone in hexane) and aluminium oxide (25Yo acetone in hexane). Spots Nos 4 and 7 were identified respectively as isocryptoxanthin and 4-hydroxy-4'-keto-fl-carotene, since they migrate together with the corresponding authentic samples in thin-layers of silica gel G and aluminium oxide using as solvent 25~o acetone in hexane. Carotenoids in R. americana midgut cells Midgut cells, homogenized in 0.1 M NaCI at neutral pH and centrifuged at 10,000g for 10min, provide a pale yellowish supernatant whose visible absorption spectrum has not discernible peaks. Nevertheless, midgut carotenoids amount to 0.89 #g/ midgut corresponding to 37Yo of the total present in the whole larvae (2.4#g/larva). These data suggest that in midgut carotenoids are largely not proteinbound. Thin-layer chromatography of midgut carotenoids is showed in Fig. 1 and their quantification is displayed in Table 1. Isolation and properties of the R. americana haemolymph yellow lipoprotein As a first attempt to isolate the chromoproteins, R.

494

W.R. TERRA,C. FERREIRAand A. G. DE BIANCHI c~ 6

L

r'; y C)

V c

V

d

I

0

015

1 Rm

0

0.5

1 Rm

Fig. 2. E]ectrophoreti¢ separation in 7~ polyacrylamide gel of proteins from unfractionatcd haemolymph and from haemolymph sediments obtained in several ranges of ammonium sulfate saturation. (a) unfractionated haemolymph; (b) 50-60~o; (c) 60-70~o; (d) 70-80~; y, yellow lipoprotein; V, violet carotenoprotein; L, lemon pigment.

americana haemolymph were fractionated by a stepwise saturation (50, 60, 70 and 80~) with ammonium sulfate. The relative amounts of proteins (in parentheses) found in the different fractions were: 0-50Vo (61.7~), 50-60~ (16.4~), 60-70~o (8.7~o), 70-80~ (5.1 ~o), remaining supernatant (8.1~o). Proteins saltingout at 50% ammonium sulfate saturation were poorly colored and hence they were discarded. The other fractions were analyzed electrophoretically and the data showed (Fig. 2) that proteins precipitating at saturations higher than 60~ are mainly the yellow and violet chromoproteins. Haemolymph proteins precipitating in the range of 60-80~o ammonium sulfate saturation were passed through a column of DEAE-Sephadex equilibrated with 0.05 M phosphate buffer pH 6.0. The yellow chromoprotein was not retained in the column, being eluted out free from the violet chromoprotein, although contaminated by non-colored proteins, as assured by polyacrylamide gel electrophoresis. The yellow eluate was then retained in a CMC-Sephadex column equilibrated with 0.05 M phosphate buffer pH 6.0, from which it was removed with 0.05 M phosphate buffer pH 6.0, 0.1 M NaC1. A 7~ polyacrylamide gel electrophoresis of the eluate showed the existence of two yellowish protein bands, in contrast to the only one seen in unfractionated haemolymph, suggesting the chromoprotein dissociates or becomes more labile after CMC-Sephadex chromatography. Due to that, the yellow chromoprotein molecular weight obtained by electrophoresis of unfractionated haemolymph (287,000) was considered to be that one of the native protein. Purified yellow chromoprotein results in two polypeptide bands after SDS-

polyacrylamide gel separation. Those bands correspond to haemolymph peptides No. 1 and 2, whose molecular weights, determined according to Frank & Rodbard (1975), were respectively 176,000 and 101,000 (De Bianchi, 1977). The data suggest the native yellow chromoprotein (molecular weight 287,000) is a dimer composed to two subunits whose molecular weights are 176,000 and 101,000. The study of the absorption spectrum and of the carotenoids of the yellow chromoprotein was carried out in the preparation obtained after DEAESephadex chromatography of haemolymph chromoproteins. Although after CMC-Sephadex chromatography the yellow chromoprotein seems to be homogeneous, the fact it is dissociated and considerably bleached makes this preparation inappropriate for those purposes. The absorption spectrum of the yellow chromoprotein does not change in denaturating conditions (0.1 N HC1 or 0.1 N NaOH) and it is similar to the one of its carotenoids, although bathochromically shifted (Fig. 3). Boiling could not be used as denaturating agent in this study because the protein completely coagulated on heating. The carotenoids extracted from the yellow chromoprotein was separated in thin-layer of silca gel G (Fig. I), identified and quantitatively evaluated (Table 1). Haemolymph carotenoids in three species of Rhynchosciara Table l shows that the relative concentrations of haemolymph carotenoids vary considerably among different species of Rhynchosciara which together with

Carotenoids from Rhynchosciara

495

i

0.4

t~

~

0

.

2

300

400

500

600

A,nm

Fig. 3. Absorption spectra of the DEAE-Sephadex purified yellow chromoprotein and of its carotenoids. ( ) yellow chromoprotein in 0.05 M phosphate buffer pH 6.0; ( - - ) carotenoids extracted from the yellow chromoprotein. their differences in absolute contents (Table 2) determine the color of their haemolymphs. The bright red Rhynchosciara sp haemolympb has the higher content of carotenoids, with predominance of keto carotenoids, while the pale yellow haemolymph of R. milleri has a low content of carotenoids with less ketocarotenoids. The red haemolymph of R. americana has an intermediate content of total carotenoids and of ketocarotenoids, which increases with the age of the larvae (Table 2). The differences found in carotenoid contents among the larvae of Rhynchosciara seem to be the result of the haemolymph titres of the yellow and violet chromoproteins. This assumption is based on the observation that protein electropherograms of the haemolymph of the larvae of R. americana, Rhynchosciara sp and R. milleri, at different ages, display the same chromoproteins as shown in Fig. 2a, although at different concentrations. DISCUSSION

The major carotenoids found in R. americana haemolymph were echinenone, canthaxanthin, cryptoxanthin and fl-carotene; minor carotenoids were isocryptoxanthin and 4-hydroxy-4'-keto-fl-carotene (Table 1), The presence of cis-4-keto-fl-carotene in the haemolymph was considered to be an artefact. Since among the haemolymph carotenoids only fl-carotene is also present in Rhynchosciara food (Terra et al., 1976), the other carotenoids must be derived from the dietary fl-carotene by oxidation. Fig. 4 display the

proposed oxidative pathways of carotenoids in R. americana larvae basing on the carotenoids found in the haemolymph and on purified chromoproteins. Data on the violet chromoprotein came from Terra et al. (manuscript in preparation), Three color mutants are known in R. americana larvae from which two were recognized as being metabolic disorders affecting carotenoid metabolism (Terra et al., 1976). LI-mutant does not display any haemolymph protein-bound carotenoid and was considered to be a consequence of mutation in step 1 (Fig. 4). LII-mutant does not display the violet chromoprotein, although the apoprotein is present (Terra et al., 1976). According to Fig. 4 such phenotype should arise if mutation has occurred in steps 2 or 3. Mutation only on steps 4 or 5 should produce a chromoprotein with altered color, which was not found (cf. Terra et al., 1976), since echinenone alone is capable of binding the violet chromoprotein apoprotein (Terra et al., manuscript in preparation). The site of carotenoid oxidation in R. americana larvae is not known with certainty, although we may infer it should be in the midgut. This follows from the finding that midgut is the only tissue which has measurable amounts of carotenoids besides haemolymph. Carotenoids are absent from fat body up to the spinning stage, when it becomes increasingly colored by carotenoids (unpublished observations). Furthermore all the major haemolymph carotenoids were found in midgut in large amounts. The fact fl-carotene is the major midgut carotenoid (Table 1) should be a consequence of the storage of the ab-

Table 2. Haemolymph carotenoid contents (pg/ml) in larvae of different species and ages* Rhynchosciara sp

R. milleri

(Fourth instar)

(Fourth instar)

Instar

R. americana

Age (days)

/~g/ml

121

45

Third Fourth Fourth

25 33 45

21 24 76

* Carotenoid contents were calculated assuming Ei~m = 2500 for all carotenoids. Rhynchosciara sp and R. milleri fourth instar larvae were collected in the nature. They were fully grown larvae and should be compared with R. americana 45 days

old.

W.R. TERRA,C. FERREIRAand A. G. DE BIANCHI

496

/5'-carotene

dietary

,L'

incorporated

YELLOW

fi- carotene

OH i socryptoxanthin

/

cryptoxanthin

~

CHROMOPROTEIN

0

0

echinenone

VIOLET CHR OMOPROTEIN

OH

4 - hydroxy- 4'- keto-fl-carotene

0 cant haxanthin

Fig. 4. Possible pathways in the metabolism of carotenoids in R. americana. Numbering correspond to the different steps in absorption and oxidation of dietary fl-carotene. sorbed dietary carotenoid before its oxidation to the different xanthophylls. As commented before, an aqueous extract of the midgut does not show discernible absorption peaks with visible light. This suggests the carotenoids are not dispersed in the aqueous fraction of the midgut cells complexed with proteins, but should be associated with the oil droplets visible inside the cells under the phase constrast-microscope. In this connection it is interesting to recall midgut cells store considerable amounts of lipids which correspond to 22~o of their dry weights (Terra et al., 1975). At this point an important question arises: how carotenoids, presumed to be oxidized in midgut, are loaded onto haemolymph proteins which are synthesized in the fat body in several insects (Chen, 1978) and for which there is evidence that the same occurs in R. americana (De Bianchi, 1977)? The yellow liproprotein certainly binds carotenoids together with the lipids it transports directly from the midgut (see below). The violet carotenoprotein, which is not a lipoprotein (Terra et al., manuscript in preparation), also must take carotenoids directly from the midgut, since the alternative possibility, i.e. the binding of carotenoids taking place in the fat body, it is discarded by the finding of a mutant (O-mutant, Terra et al., 1976) which possess as carotenoprotein only the violet chromoprotein. In this mutant, there is no way to transport carotenoids from the midgut to the fat body, since in R. americana all the carotenoids are protein-bound (Terra et al., 1974). Oxidative pathways of dietary//-carotene similar to the one proposed here seem to occur in some insects such as several Coleoptera species (e.g. Leuenberger & Thommen, 1970; Mummery & Valadon, 1974). Nevertheless, as much as we know, there is no secure information at which tissue that oxidation steps

occur. Otherwise a great number of Lepidoptera species possess only carotenoids derived directly from their food, being unable to accomplish even minor alterations in the dietary carotenoids (Feltwell & Rothschild, 1974). Insects usually display several haemolymph lipoproteins, from which the major one are the chief physiological lipid carrier and it is also capable of binding juvenile hormone (Gilbert & Chino, 1974; Agosin, 1978). In some insects this lipoprotein also transports carotenoids (Gilbert & Chino, 1974). The yellow lipoprotein, which is the major lipoprotein in R. americana haemolymph (de Bianchi & Terra, 1976), should have the same functions as described above for the major lipoprotein in other insects. This follows from the observation that in its absence, as a consequence of a mutation, the larvae and adults become much smaller than the wild-types (Terra et al., 1976). Furthermore its structure is similar to the major lipoprotein from several insects, which is composed of a large and a smaller protein subunit associated with lipids (cf. Pattnaik et aL, 1979). Carotenoids seem to be freely solubilized in the lipids associated to the R. americana yellow lipoprotein, since only a little bathochromic displacement is observed in their visible absorption spectra when they are binded to the protein (Fig. 3). Such displacements can be accounted for by differences in the refractive index of the material in which carotenoids are solubilized in the lipoprotein and of hexane (cf. Zagalsky, 1976). Acknowledoements--This work was supported by grants from the Funda~.o de Amparo ~ Pesquisa do Estado de S~o Paulo (FAPESP). We are much indebted to the Brazilian branch of the Hoffman La Roche, Switzerland, for a gift of authentic samples of carotenoids and to Miss L. Y.

Carotenoids from Rhynchosciara Nakabayashi for technical assistance. C. F. is a graduate fellow of FAPESP.

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