A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.     

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.     

Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism

Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer

In this study we report the purification and characterization of a lipid transfer particle (LTP) from Rhodnius prolixus hemolymph, and its participation in phospholipid and diacylglycerol transfer processes. (3)H-diacylglycerol labeled low density lipophorin from Manduca sexta ((3)H-LDLp) was incubated with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. Following incubation and isolation, both lipoproteins showed equivalent amounts of (3)H-labeled lipids. Hemolymph was subjected to KBr gradient ultracentrifugation. SDS-PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP containing fractions were assayed and lipid transfer activity was observed. Purification of LTP was accomplished by (i) KBr density gradient ultracentrifugation, (ii) size exclusion, (iii) Cu(++) affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated approximately 770 kDa, comprising three apoproteins, apoLTP-I (315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Phospolipid content of (32)P-LTP was determined after two-dimensional TLC. (32)P-phospholipid-labeled and unlabeled lipophorins, purified from R. prolixus were incubated in the presence of LTP resulting in the time-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process.     

Structural studies of lipophorin in insect blood by differential scanning calorimetry and 13C nuclear magnetic relaxation measurements. Location of hydrocarbons

The possible structure of lipophorin in insect blood (hemolymph) was investigated by differential scanning calorimetry (DSC) and 13C nuclear magnetic relaxation studies. The DSC heating curves of intact lipophorins showed endothermic peaks between -3 and 40 degrees C for lipophorins which contain hydrocarbons, whereas no such peaks were observed for lipophorins which do not contain this lipid. Hydrocarbon fractions isolated from the lipophorins showed endothermic peaks similar to those obtained from intact lipophorin in terms of the transition temperatures, the shapes, and the enthalpy changes. 13C spin lattice relaxation times of the (CH2)n resonance of hydrocarbons of intact lipophorin were measured as a function of temperature and revealed that the motions of hydrocarbon chains changed coincidentally with the onset and offset of phase transition. These data suggest the presence of a hydrocarbon-rich region within the lipophorin particles.     

Structure of apoproteins in insect lipophorin

The two polypeptide chains of cockroach and locust lipophorins were separated and their amino acid compositions were determined. Circular dichroic spectra of the lipophorins and apolipophorin from 190 to 250 nm showed a single trough at 218 nm and a peak at 194 nm. Infrared spectra of the lipophorins in D2O showed a strong peak at 1625 cm-1 and a weak shoulder at 1693 cm-1 corresponding to v (pi, 0) and nu (0, pi) of antiparallel pleated sheet. The resonance frequency splitting delta nu = nu (0, pi) -nu (pi, 0) was 68 cm-1, which was larger than that of ordinary globular proteins containing antiparallel pleated sheet. From circular dichroic and infrared spectra it was concluded that lipophorins contained polypeptides rich in antiparallel pleated sheet with longer unbroken extensions than the case for ordinary globular proteins. Partial proteolytic digestion study of lipophorins with trypsin, chymotrypsin, and subtilisin showed that the larger apolipophorin (AL1) was exposed to the surface of the particle and the smaller apolipophorin (AL2) lay protected from the attack of the enzymes. Crosslinked products between AL1 and AL2 were readily obtained when dimethylsuberimidate or dimethyladipimidate was added to the lipophorin solution, without giving lipophorin dimers, suggesting that the two chains were located within 11 A from each other. Such structural features of insect lipoprotein were compared with other insect lipophorins and the human serum low-density lipoprotein (LDL). Similarities between lipophorins and LDL were found in the molecular weight, amino acid compositions, and the secondary structure of major apoproteins.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

A discoidal lipoprotein from the coelomic fluid of the polychaete Nereis virens

A discoidal lipoprotein was isolated from the coelomic fluid of the polychaete, Nereis virens, by density gradient centrifugation. The lipoprotein was present in both sexes and moved as a uniform band in an agarose gel. The average diameter of the lipoprotein particles determined by electron microscopy was 42 nm with a thickness of 10 nm. SDS electrophoresis showed two apoprotein subunits with molecular masses of 247 and 85 kDa, respectively. In lectin blots, both apoproteins were reactive with Concanavalin A indicating the presence of N-glycans. The small subunit was also reactive with peanut lectin, indicating additional O-glycosylation. The total lipid content was 48% and consisted mainly of phospholipids and some diglycerides as judged by thin layer chromatography. The estimated native molecular mass of N. virens lipoprotein ( approximately 675 kDa) lies in the range of vertebrate high-density lipoprotein and insect lipophorins. The size of the apoproteins is similar to those found in insects, while the composition of the lipid fraction is more similar to that of crustacean lipoproteins.     

Isolation and characterization of lipophorin from Drosophila melanogaster larvae

The hemolymph lipoprotein lipophorin has been isolated from third-instar Drosophila melanogaster larvae by a technique that involves homogenization of whole larvae in a medium containing protease inhibitors and purification of the lipoprotein by density gradient centrifugation. Drosophila lipophorin has a density of 1.16 g/ml and is composed of 62.5% protein, 23.1% phospholipid, 7.4% diacylglycerol, 5.4% triacylglycerol, 0.9% hydrocarbon, and 0.7% sterol. As is the case with other insect lipophorins, Drosophila lipophorin contains two apolipoproteins, apolipophorin-I (M_r ≈ 275,000) and apolipophorin-II (M_r ≈ 76,000). Drosophila apolipophorin-I does not crossreact with antibodies prepared against apolipophorin-I from Manduca sexta.     

Lipophorin lipase from the yolk of Manduca sexta eggs: identification and partial characterization.

In the hawkmoth Manduca sexta high density lipophorin from adult insects (HDLp-A) delivers lipids to developing oocytes. During this lipid delivery HDLp-A is taken up by the oocyte and converted to a very high density lipophorin (VHDLp), which is stored in protein storage granules (yolk bodies). A membrane-free lysate of isolated M. sexta yolk bodies was demonstrated to contain lipoprotein lipase activity that hydrolyses the diacylglycerol of HDLp-A. With HDLp-A as a substrate yolk body lipophorin lipase (YBLpL) activity was shown to be maximal between pH 9 and pH 9.5. NaCl concentration was optimal between 0.7 M and 1 M. YBLpL activity required neither bovine serum albumin nor calcium ions but appeared to be stimulated by 5 mM EDTA. Diisopropyl fluorophosphate effectively inhibited YBLpL activity, indicating the presence of a serine in the active site of the enzyme. The identified lipase activity co-eluted with lipophorins and vitellins from the yolk in the void volume of a Sephadex G-75 gel filtration column. This observation suggests that the lipase has a Mr of more than 80,000, or that the enzyme is associated with the lipoproteins. Incubation of HDLp-A with yolk body lysate converted HDLp-A to two classes of higher density lipophorins. The highest density lipophorins produced during this incubation approached the density of VHDLp as it is isolated from mature eggs. The possible role of YBLpL activity in the delivery of lipids to developing oocytes is discussed.     

31P-NMR study of the phospholipid moiety of lipophorin subspecies.

31P-NMR spectra of four distinct subspecies of Manduca sexta hemolymph lipophorin revealed the presence of two resonances separated by 0.6 ppm. Phospholipid analysis of the lipoproteins showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present and their mass ratio correlated well to the intensity of the two resonances in each of the different subspecies. The two resonances persisted in 31P-NMR spectra of organic solvent extracts of lipophorin. These results, together with the fact that PE, but not PC, can form an intramolecular hydrogen bond between the phosphate oxygen and the amino group of ethanolamine, resulting in deshielding of the phosphorus nucleus (and a 0.6 ppm downfield shift), strongly suggest the resonances observed represent the PC and PE components of these lipoproteins. 31P-NMR line-width data obtained as a function of temperature and solvent viscosity were used to calculate the chemical shift anisotropy (delta sigma), intrinsic viscosity (eta'), and lateral diffusion coefficients (DT) of PC and PE in different lipophorin subspecies. eta' and DT for PC and PE were similar among high-density lipophorins but differed in low-density lipophorin (LDLp). These differences may be related to the large increase in diacylglycerol content in this particle and/or the association of up to 16 molecules of apolipophorin III. On the basis of the known lipid compositional differences between LDLp and high-density lipophorin subspecies, we propose that uptake of large amounts of diacylglycerol during LDLp formation results in partitioning of this lipid to the surface monolayer where it intercalates between phospholipid molecules. Diacylglycerol intercalation creates gaps between phospholipid head groups that expose the hydrophobic surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

Lipid circulation in spiders. Transport of phospholipids,free acids and triacylglycerols as the major lipid classes by a high-density lipoprotein fraction isolated from plasma of Polybetes pythagoricus

A high-density lipoprotein (HDL) fraction was isolated from the hemolymphatic plasma of the spider Polybetes pythagoricus by density-gradient ultracentrifugation. Hydrated density (1.13 g/ml), electrophoretic mobility (SDS—PAGE) of apoproteins and lipid classes composition were determined. Lipids were identified by HP-TLC and auxiliary techniques; they were quantified by TLC-FID. The protein moiety is composed of two main apoproteins (250 and 76 kDa, respectively) and several polypeptides of low molecular weight. It resembles the apoliphorins of insects and some other arachnids. The lipid composition differs from most lipophorins. Phospholipids amount to more than 60% of total lipids, while diacylglycerols (2.4%) are supplanted by triacylglycerols (16.5%) as the main circulating energetic lipids.     

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.     

Rhodnius prolixus LIPOPHORIN: LIPID COMPOSITION AND EFFECT OF HIGH TEMPERATURE ON PHYSIOLOGICAL ROLE

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin‐layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3‐min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.     

Role of lipophorin in lipid transport to the insect egg

Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total egg lipid is derived from de novo synthesis by the follicles. The remaining egg lipid originates in the fat body and is transported to the ovary by lipoproteins. Vitellogenin, the major egg yolk lipoprotein, accounts for 5% of the total egg lipid. The remaining 95% lipid is attributable to the hemolymph lipophorins, adult high density lipophorin (HDLp-A) and low density lipophorin (LDLp). When HDLp-A that is dual labeled with 3H in the diacylglycerol fraction and 35S in the protein moiety is incubated with follicles in vitro, the ratio of 3H:35S in the incubation medium does not vary and is similar to the ratio of the labels that are associated with the follicles. In an accompanying paper (Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) J. Biol. Chem. 263, 8740-8747), we show that HDLp-A is sequestered by the follicles without subsequent hydrolysis of its apoproteins. These results, together with those presented in this paper, support our conclusion that HDLp-A is not recycled back into the hemolymph after it is internalized by the follicles and, therefore, does not function as a reusable lipid shuttle between the fat body and the ovary. When follicles are incubated with dual labeled LDLp, the diacylglycerol component of the particle is internalized by the follicles without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by which lipid is delivered to the ovary.     

Lipophorin density variation during oogenesis in Rhodnius prolixus

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations of lipophorins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophoin of different densities. Arch. Insect Biochem. Physiol. 35:301-313, 1997.© 1997 Wiley-Liss, Inc.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.