Synthesis and release of hydrocarbons by the oenocytes of the desert locust, Schistocerca gregaria

When incubated in vitro, the oenocytes in the peripheral fat body of the desert locust incorporate Na-¹⁴C-acetate into hydrocarbons (paraffins). The presence of haemolymph in the incubation medium greatly stimulates the release of the ¹⁴C-hydrocarbons into the medium. The labelled hydrocarbons appear to be rapidly released by the cells into the incubation medium as a function of time provided that haemolymph is present. The fact that the oenocytes not only synthesize ¹⁴C-hydrocarbons but also release them into the medium supports the hypothesis that the oenocytes of the desert locust synthesize cuticle lipids.     

Characterization of Colorado potato beetle lipophorin: A hydrocarbon-rich diacylgycerol-poor lipophorin

• 1.1. The lipid composition of lipophorin from the Colorado potato beetle, Leptinotarsa decemlineata Say, was analyzed.
• 2.2. This insect lipophorin contains 44% lipid and is characterized by large amounts of hydrocarbons and small amounts of diacylglycerol.
• 3.3. This is the first observation of a diacylglycerol-poor insect lipophorin in haemolymph.
• 4.4. Since the main energy source for flight in the Colorado potato beetle is proline, the low diacylglycerol content in lipophorin must be related to its peculiar flight metabolism.
• 5.5. This lipophorin, however, can still take up appreciable amounts of diacylglycerol from the locust fat body. Hydrocarbon uptake by this lipophorin was also demonstrated.
• 6.6. The main function of this lipophorin therefore seems to be transport of hydrocarbons from oenocytes to the cuticle.

     

Adipokinetic hormone causes formation of a low density lipophorin in the house cricket,Acheta domesticus

In the house cricket, Acheta domesticus, injection of adipokinetic hormone induces the formation of a low density lipophorin via uptake of diacyglycerol from the fat body. Cricket low density lipophorin contains apolipophorin-III, which was purified and partially characterized. Cricket apolipophorin-III has a molecular weight of about 18,000, is not glycosylated, and has an isoelectric point of 4.8. Its amino acid composition is more similar to apolipophorin-III from Locusta migratoria than to that from Manduca sexta. The amino terminal sequence of cricket apolipophorin-III shows only limited homology to the amino terminal sequences of apolipophorin-III from L. migratoria and M. sexta.     

Lipophorin transport of hydrocarbon during early vitellogenesis in the silkworm,Bombyx mori

Lipophorin transports the hydrophobic molecule from origin to destination in time specific manner. Here, we investigate the hydrocarbon composition in the lipophorin during day 2 of pupal silkworm, Bombyx mori. Lipophorin was isolated from hemolymph by immunoprecipitation; lipid extracted and analyzed in GC-MS, resulting in seventeen compounds including eight hydrocarbons. Which were searched with the Pherobase (Pheromone database) and functional roles analysed. All hydrocarbons denoted as a pheromone except pentatriacontane in lepidopteron insects. Other hand, hydrocarbons such as hexadecane, nonadecane, undecane and hexacosane specifically refer as an attractant, additionally heptacosane, octacosane and docosane indicate as an allomone in non-lepidopteron insects. As well as, docosane and undecane perform as kairomone. Identified pheromone interaction with lipophorin was analysed by molecular docking and shows the best binding score. All hydrocarbons bound in a lipid binding pocket, most of these sharing the same binding sites for hydrophobic interaction and hydrogen bonding. Overall findings elucidated that lipophorin associated with different hydrocarbons by hydrogen bonds and hydrophobic interactions, and each of the hydrocarbon having various functional roles.     

Synthesis and release of lipophorin in larvae of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The source of the lipophorin present in the larval haemolymph of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Although lipophorin was shown to be one of several proteins released from cultured fat body and midgut, only fat body was shown to synthesize lipophorin. Fat body, incubated in a medium containing [³H]leucine, was shown to release radiolabelled lipophorin using immunoprecipitation. Similar studies using midguts incubated in a medium containing [³H]leucine did not reveal any synthesis of lipophorin. Lipophorin was isolated by density-gradient ultracentrifugation from media in which the fat bodies of about 600 diapausing larvae had been incubated for 4 hr. The isolated lipophorin had a peak density of 1.11 g/ml, and contained various lipids including diacylglycerol, triacylglycerol, sterol, hydrocarbon, free fatty acid, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin.     

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.Download video file.^{(173M, mp4)}     

Characterization of termite lipophorin and its involvement in hydrocarbon transport

The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae.     

In vitro synthesis and secretion of lipophorin by the fat body of nondiapause and prediapause larvae of the southwestern corn borer,Diatraea grandiosella

The capacity of the fat body of nondiapause, prediapause and diapause larvae of the southwestern corn borer, Diatraea gradiosella, to synthesize and release lipophorin was examined in vitro using [³H]leucine as the radiotracer. Synthesis and release of [³H]lipophorin by the fat body peaked in 11–13 day-old fifth instar nondiapause larvae, which coincided with their feeding period. The rate of lipophorin synthesis in the fat body of newly ecdysed pupae was extremely low. Synthesis and release of [³H]lipophorin by the fat body of prediapause larvae occurred at the highest rates in 20–35 day-old fifth and sixth instars, and declined to virtually undetectable levels after larvae entered diapause around 40 days-of-age. Immunoprecipitation of [³H]lipophorin from fat body of 13 day-old nondiapause larvae that had been pulse-labeled with [³H]leucine showed that the half life of lipophorin synthesis and processing was about 40 minutes. Release of total protein and lipophorin from the fat body of 13 day-old nondiapause larvae into Grace's medium was inhibited by 56 and 60%, respectively, when 10 μg/ml tunicamycin was incorporated into medium.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Purification of adult Drosophila melanogaster lipophorin and its role in hydrocarbon transport

Lipophorin was isolated from homogenized adult Drosophila melanogaster. It is stained by Sudan Black and has a native molecular mass of 640 kD and a density of 1.12 g/ml. It consists of two glycosylated apoproteins of 240 and 75 kDa. Gas chromatography and mass spectrometry showed that lipophorins isolated separately from virgin 3-day-old male and female flies were associated with specific hydrocarbons, and that these were the same hydrocarbons found in male and female cuticles, respectively. Moreover, a pool of internal hydrocarbons was demonstrated for the first time, with chain lengths similar to those of the cuticular pool. Studies on the fate of the hydrocarbons synthesized de novo after topical applications of radiolabelled fatty acid precursors showed a decrease of the internal pool of hydrocarbons with time, concomitant with an increase of the cuticular pool. These results suggest that hydrocarbons synthesized at an internal site, possibly in oenocytes, may be transported to the cuticle of the flies by lipophorin. © 1996 Wiley-Liss, Inc.     

Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.     

Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro

Isolated prothoracic glands from Tenebrio larvae synthesize in vitro α-ecdysone, but not β-ecdysone from 4-¹⁴C-cholesterol. Isolated abdominal oenocytes from the larvae synthesize mainly β-ecdysone, but only little α-ecdysone. When prothoracic glands and oenocytes are cultured together, the α-ecdysone derived from the prothoracic glands is oxidized by the oenocytes to β-ecdysone. The newly synthesized hormones are not stored in the cells, but are secreted into the medium if sufficient amounts of non-labelled hormones are present. If no unlabelled hormones are added to the culture medium, the newly formed hormones are converted to a large extent into polar conjugates.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.     

Lipoprotein lipase activity in the flight muscle of Locusta migratoria and its specificity for haemolymph lipoproteins

Lipoprotein lipase activity in flight muscle homogenates of Locusta migratoria was measured, using natural radiolabelled lipoproteins as substrates. The flight specific lipoprotein A⁺ (or low density lipophorin) stimulated lipoprotein lipase activity several-fold compared to the resting lipoprotein A_y (or high density lipophorin). However, with the high mol. wt lipoprotein fraction O_AKH as a substrate, lipase activity was even doubled compared to lipoprotein A⁺. Lipase activity was not increased in flight muscle homogenates of insects which had flown. Neither adipokinetic hormone, nor octopamine had any direct effect on lipoprotein lipase activity. Aspects of hormonal regulation and apoprotein activation of the locust flight muscle lipoprotein lipase are discussed and compared with the model for vertebrate lipoprotein lipase.     

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⁺.     

Food intake in Blattella germanica (L.) nymphs affects hydrocarbon synthesis and its allocation in adults between epicuticle and reproduction

The causal relationship between food intake and hydrocarbon synthesis was examined in vivo and in vitro. Fed Blattella germanica (L.) nymphs synthesized hydrocarbons in a stage‐specific manner, with high rates occurring in the first 6 days of a 13‐day last stadium, in relation to feeding. A similar pattern was exhibited in vitro by sternites and tergites from fed nymphs. In contrast, starved nymphs synthesized hydrocarbons at normal rates for the first 2 days, but then synthesis declined and ceased by day 6. Their abdominal sternites and tergites displayed a similar biosynthetic pattern in vitro, showing that starved tissues lost the capacity to synthesize hydrocarbons, even when provided appropriate nutrients. Synthesis resumed within 2 days of being fed on day 6, reaching a maximum rate 6 days later. Some hydrocarbon appeared on the nymphal cuticle, but almost 4‐fold more hydrocarbon was internal in hemolymph lipophorin, fat body, and the developing imaginal cuticle. Because most hydrocarbon synthesized in nymphs provisions the adult, and synthesis is related to food intake, we examined trade‐offs in allocations in food‐limited insects. Nymphs provided with insufficient quantities of food allocated normal amounts of hydrocarbons to the nymphal epicuticle, but molted into smaller adults with significantly less internal hydrocarbons. These cockroaches directed nearly normal amounts of hydrocarbons to their epicuticle, oocytes, and oothecae, at the cost of internal hydrocarbon reserves for repair and subsequent gonotrophic cycles. Hydrocarbons, thus, appear to serve an important cross‐stadial resource and the object of competition among several nymphal and adult tissues. Arch. Insect Biochem. Physiol. 41:214–224, 1999. © 1999 Wiley‐Liss, Inc.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse-labeling of lipophorin with [³⁵S]methionine showed that apolipophorin-I and -II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse-labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m-chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin-I or -II. Incubation of fat bodies with [³H]oleate resulted in the secretion of lipophorin containing [³H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [³H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [³H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin-associated lipids and secretion of lipids mediated by the lipid-transfer particle outside the plasma membrane of the fat body.     

Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni

Protein synthesis at various times during the pregnancy cycle of G. austeni was determined by autoradiographic measurement of the incorporation of H³-leucine and H³-tyrosine into the cells of the fat body, oenocytes, milk gland and epidermis. The rate of utilization of these molecules is such that the labelled pool in the haemolymph is depleted before 0.5 hr after injection. The incorporation of both amino acids into fat body and oenocytes is high at eclosion and just after larviposition, with the incorporation of tyrosine by the oenocytes being much higher than that in the fat body. The same pattern of incorporation is observed in the epidermal cells. Label also appears in the endocuticle during the first 10 days of adult life. Except during the first 4 days following emergence, the incorporation of the two amino acids into the milk gland is very high, with periods of less intense protein synthesis at about the time of larviposition. The milk gland represents a highly efficient secretory system, with a t₅₀ of less than 30 min.