Lipophorin of the larval honeybee, Apis mellifera L

Most insects have a major lipoprotein species in the blood (hemolymph) that serves to transport fat from the midgut to the storage depots in fat body cells and from the fat body to peripheral tissues. The generic name lipophorin is used for this lipoprotein. In larvae of the honeybee, Apis mellifera, a lipophorin has been found with properties that correlate well with those of the only other lipophorin reported for an immature insect, that of the tobacco hornworm, Manduca sexta. The honeybee lipophorin (Mr = 530,000) has a density of 1.13 g/ml, contains approximately 41% lipid and 59% protein, and contains two apoproteins, apoLp-I, Mr = 250,000 and apoLp-II, Mr = 80,000, both of which are glycosylated. The lipids consist predominantly of polar lipids, of which phospholipids and diacylglycerols represent 60% of the total. When the intact lipophorin is treated with trypsin, apoLp-I is rapidly proteolyzed, while apoLp-II is resistant, indicating a difference in exposure of the two apoproteins to the aqueous environment. Honeybee apoLp-II cross-reacts with antibodies to M. sexta apoLp-II, but not to anti-M. sexta apoLp-I. No cross-reactivity of honeybee apoLp-I to anti-M. sexta apoLp-I was observed.     

Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust

Spleen lymphocytes from mice immunized with locust native low-density lipophorin A⁺ (LDLp) were fused with nonproducing myeloma cells, strain Sp 2/0. Hybridomas that were isolated from the fused cells produced antibodies specific for LDLp and the high-density lipophorin A_yellow (HDLp). Monoclonal strains were generated through cloning by limiting dilution from those hybridomas synthesizing antibodies specific for apolipophorins (apoLp)-I, -II, and -III of LDLp. Additionally, a hybridoma strain that was obtained after fusion of lymphocytes from mice immunized with apoLp-III produced antibodies that bind to apoLp-III and native LDLp. Some features of LDLp and HDLp were studied using these antibodies. It could be demonstrated that apoLp-I and apoLp-II are not immunochemically identical and are exposed in the native particle of both LDLp and HDLp. It was also shown that in both lipophorins apoLp-II is less exposed than apoLp-I, whereas in LDLp apoLp-III is mainly exposed; some apoLp-III could also be detected in HDLp. Tween-20, a nonionic detergent, appears to affect the binding of anti-apoLp-I, -II, and -III to both LDLp and HDLp. The monoclonal antibodies specific for locust apolipophorins do not bind to the respective apoproteins of lipophorins from other insects.     

Biosynthesis and secretion of insect lipoprotein.

Biosynthesis of high density lipophorin (HDLp) was studied in larvae and adults of the migratory locust, Locusta migratoria. In an in vitro system, fat bodies were incubated in a medium containing a mixture of tritiated amino acids. Using SDS-PAGE and immunoblotting, it was shown that larval and adult fat bodies secreted both HDLp apoproteins, apolipophorin I (apoLp-I) and apolipophorin II (apoLp-II). Radiolabel was recovered in both apoproteins, indicative of de novo synthesis. The density of the fractions containing the apoproteins synthesized and secreted by larval and adult fat bodies was determined by density gradient ultracentrifugation. A radiolabeled protein fraction was found at density 1.12 g/ml. Using an enzyme-linked immunosorbent assay for detecting apoLp-I and apoLp-II, it was demonstrated that both apoproteins were present in this fraction, which had a density identical to that of circulating HDLp in hemolymph. Lipid analysis revealed that it contained phospholipid, diacylglycerol, sterol, and hydrocarbons. From these results it is concluded that the fat body of the locust synthesizes both apoLp-I and apoLp-II, which are combined with lipids to a lipoprotein particle that is released into the medium as HDLp.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Manduca sexta lipid transfer particle: Synthesis by fat body and occurrence in hemolymph

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins. © 1996 Wiley-Liss, Inc.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Binding of locust high-density lipophorin to fat body proteins monitored by an enzyme-linked immunosorbant assay

Binding of locust high-density lipophorin (HDLp) to fat body proteins coated on immunoassay plates was studied using the ELISA method and ligand blotting techniques. HDLp binding proved to be correlated with the amount of fat body protein coated. From the concentration-dependent total HDLp binding an equilibrium dissociation constant could be calculated (Kd = 1.6 x 10(-8) M). Heparin inhibits the HDLp binding, indicating that positively charged groups are involved in the HDLp-fat body interaction. These groups were shown to be arginyl residues, as the arginine-specific treatment of HDLp by 1,2-cyclohexanedione resulted in a approximately 50% decrease in the binding ability of HDLp. HDLp binding is also affected by the pH. A decrease from pH 7.5 to pH 6.5 increases the binding affinity by approximately 250%. A monoclonal antibody specific for apolipophorin-II (apoLp-II) hampered the HDLp binding significantly, whereas a monoclonal anti-apoLp-I had no effect. Locust fat body HDLp binding proteins are highly specific for locust HDLp.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Structural studies on lipophorin, an insect lipoprotein

An insect high density lipoprotein, lipophorin, can be rapidly isolated from larval Manduca sexta (tobacco hornworm) hemolymph by single vertical spin density gradient ultracentrifugation. The two apolipoproteins (Mr = 245,000 and 78,000; designated apoLp-I and apoLp-II, respectively) were readily dissociated and separated in 6 M guanidine HCl by gel permeation chromatography. ApoLp-I and apoLp-II showed no immunological cross-reactivity on electrophoretic blots of sodium dodecyl sulfate-polyacrylamide gels. ApoLp-I and apoLp-II from lipophorin of adult M. sexta behaved identically to their larval counterparts. Amino acid compositions of larval apoLp-I and apoLp-II were similar except with respect to tryptophan and cysteine; apoLp-I contained 32 residues/mol of tryptophan (1.5 mol%) and 22 residues/mol (1.1 mol%) of cysteine; apoLp-II contained 2 residues/mol of tryptophan (0.2 mol%) and 14 residues/mol of cysteine (2.1 mol%). In double immunodiffusion tests, antiserum against apoLp-I or whole lipophorin strongly precipitated lipophorin, while antiserum against apoLp-II caused only minor precipitation. This indicates relatively greater exposure of apoLp-I to the aqueous environment.     

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.     

Factors influencing cyclic AMP and diacylglycerol levels in fat body of Locusta migratoria

Forskolin (FORSK), octopamine (OA) and adipokinetic hormone (AKH) stimulate the production of diacylglycerol (DG) in fat body of the locust, Locusta migratoria and the release of DG from fat body into hemolmph. The three effectors also increase the level of cAMP in fat body, but the cAMP content is not proportional to DG production. AKH stimulates the uptake of Ca²⁺ by fat body cells and requires the presence of extracellular Ca²⁺ to increase cAMP and DG levels in fat body. The production of DG seems to be an energy-dependent process. The uptake of DG by lipophorin (LP) from fat body is also energy-dependent but does not require extracellular Ca²⁺.     

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.     

In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.

Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d approximately 1.109 g/ml) and by a low density lipophorin during flight (LDLp, d approximately 1.060 g/ml). Lipophorin presumably shuttles different lipids between sites of uptake or storage, and sites of utilization. In order to shuttle lipid, a lipid-depleted lipophorin should be able to reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d approximately 1.118 g/ml) or diacylglycerol (d approximately 1.187 g/ml) and subsequently radiolabeled in their protein moiety. Upon injection into adult moths, both particles shifted their density to that of native HDLp-A, indicating lipid loading. Also, upon subsequent injection of adipokinetic hormone, both particles shifted to a lower density (d approximately 1.060 g/ml) indicating diacylglycerol loading and conversion to LDLp. Both phospholipid and diacylglycerol loading were also studied using an in vitro system. The lipid-depleted particles were incubated with fat body that had been radiolabeled in either the phospholipid or the triacylglycerol fraction. Transfer of radiolabeled phospholipid and diacylglycerol from fat body to lipophorin was observed. During diacylglycerol loading, apoLp-III associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-III. The results show the ability of lipid-depleted lipophorins to reload with lipid and therefore reaffirm the role of lipophorin as a reusable lipid shuttle.     

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.     

In vitro release of lipophorin from the fat body of nondiapause and diapause larvae of the Southwestern corn borer,Diatraea grandiosella

The release of lipophorin and total protein was examined from the fat body of nondiapause and diapause larvae of the southwestern corn borer, Diatraea grandiosella, incubated in vitro in Grace's medium. The characteristics of the released lipophorin were compared to those of the high-density lipophorin present in the hemolymph of nondiapause and diapause larvae. Over a 4 h incubation period, the fat body of nondiapause larvae released about 1.5 times more total protein and 2 times more lipophorin per mg dry weight than did that of diapause larvae. Lipophorin isolated from the medium in which fat bodies of nondiapause and diapause larvae had been incubated and from the plasma of nondiapause and diapause larvae had similar mean densities of 1.115, 1.112, 1.117 and 1.119 g/ml, respectively. Although the lipid classes detected in lipophorin isolated from the fat body incubation medium and hemolymph were identical, more polar lipids and less diacylglycerol were associated with lipophorin isolated from fat body incubation medium then were associated with lipophorin isolated from the hemolymph. Sterols accounted for about 11% of the total lipids of lipophorin isolated from the fat body incubation medium, whereas they accounted for about 20% of the total lipids of lipophorin from hemolymph. We conclude that the fat body of feeding nondiapause larvae and nonfeeding diapause larvae releases high-density lipophorin.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

Study on the composition-structure relationship of lipophorins

High density lipophorin (HDLp is the main lipoprotein found in resting insect hemolymph. It has, in general, two molecules of apolipoproteins: apoLp-I (250 kDa) and apoLp-II (80 kDa) and a variable lipid content which ranges from 35% to 59% (w/w). Diacylglycerols (DG), phospholipids (PL), and hydrocarbons (HC) are the main lipid components, whereas cholesterol and triacylglycerols are minor components. DG content varies from 7 to 30%, PL from 11 to 24%, and HC from 0 to 15%. In order to determine the relationship between the lipid composition and the arrangement of lipid and protein components in the lipoprotein particle, a density-composition structural model was designed. The model was established by means of 12 sets of data on lipophorin density-composition relationships, and model validity was determined throughout lipoprotein space- and surface-filling conditions. Despite the differences among the lipid compositions of lipophorins, it is concluded that there are several unifying structural restrictions that govern the molecular organization of lipophorins. Quantitative treatment of the model indicates that lipophorin structure is consistent with the following. 1) Spherical particles with a protein-rich outer layer of approximately 20-21 A thickness, comprised of proteins, phospholipids, cholesterol, and small amounts of DG, and a lipid-rich core composed of HC, TG, and almost all the lipophorin DG. 2) Apolipophorins have a lipid-embedded localization within the lipoprotein particle. They might represent one of the few examples of proteins containing beta-shift structure, exerting strong hydrophobic interaction and having a lipid-embedded localization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis and secretion of insect lipoprotein: involvement of furin in cleavage of the apoB homolog, apolipophorin-II/I

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp). These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.     

Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [³H]-Cholesterol-labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent K_m of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high K_m, the high activation energy, and the lack of Ca²⁺ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.     

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.