Transport of hydrocarbons by haemolymph lipophorin in Locusta migratoria

Injection of [1-¹⁴C]acetate into the locust, Locusta migratoria, results in the incorporation of radioactivity into the hydrocarbon fraction which is subsequently released into the haemolymph in association with the lipophorin molecule. The specific capacity of the L. migratoria lipophorin to accept hydrocarbons from the oenocytes, which are believed to be the site of hydrocarbon synthesis, was demonstrated in vitro. Lipophorins from L. migratoria and Periplaneta americana displayed no specificity in their ability to accept hydrocarbons from the oenocytes of the other species. Therefore, it was concluded that the different hydrocarbon compositions of lipophorins from the two species were due to differences in the nature of the oenocytes. When lipophorin containing ¹⁴C-labelled hydrocarbon was injected into the haemocoele of L. migratoria, the labelled hydrocarbon soon appeared at the cuticular surface.The current study together with previous data support the proposal that insect lipophorin serves as a true carrier molecule for the transport of hydrocarbon from the site of synthesis (oenocyte) to the site of deposition (cuticle), in addition to its function of transporting diacylglycerol and cholesterol from the fat body and midgut.     

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.     

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.     

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.     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

Biosynthesis of very long-chain methyl-branched alcohols during pupal development in the cabbage looper,Trichoplusia ni

Internal long-chain methyl-branched alcohols and their esters in the cabbage looper, Trichoplusia ni, were synthesized exclusively during the first half of the pupal stage. Esters were synthesized beginning on day 2 post-pupation while synthesis of free alcohols reached a maximum on days 3 and 4 post-pupation. Up to 10% of the injected [1-¹⁴C]acetate and 8% of the injected [1-¹⁴C]propionate were incorporated into esters and free alcohols, while only 2% of the injected acetate and essentially no propionate were incorporated into polar lipids. Only trace amounts of labeled acetate were incorporated into acylglycerols. Upon saponification, 98% of the radioactivity incorporated into esters was found in the alcohol fraction. Both esters and alcohols were synthesized by the tissue associated with the pupal cuticle but remained internal at all times and were not deposited on the cuticle. Very low levels of fatty acid synthetase activity were found throughout pupal development.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

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.     

Biosynthesis of the hydrocarbon components of the sex pheromone of the housefly,Musca domestica L.

Biosynthesis of hydrocarbons, including components of the sex pheromone of the housefly Musca domestica L., was investigated. In vitro studies with isolated tissues from adult flies showed that the hydrocarbon components of the pheromone were synthesized primarily by the epidermal cells in abdominal segments two to seven. The incorporation of [³H or ¹⁴C]-labelled acetate, palmitate, stearate and oleate into the saturated and unsaturated hydrocarbon components showed that (Z)-9-tricosene (muscalure) was synthesized de novo by female insects and the distribution of label was consistent with a pathway in which oleic acid was elongated and then decarboxylated. A comparison of the incorporation and distribution of labelled acetate, propionate and succinate into hydrocarbons indicated that the mono- and dimethylalkanes were formed by the substitution of a methylmalonyl-CoA for malonyl-CoA during chain elongation. The incorporation of radioactivity from [1-¹⁴C]-propionate increased dramatically in female insects two days after adult emergence, which corresponds in time to the production of methyl branched alkanes. In contrast, this substrate was not efficiently incorporated at any time into male insects.     

Incorporation of labelled dietary n-alkanes into cuticular lipids of the grasshopper Melanoplus sanguinipes

Dietary hydrocarbons are incorporated into cuticular lipids of the grasshopper Melanoplus sanguinipes. Dietary secondary alcohols and ketones, however, are not incorporated into the cuticular lipids. In typical experiments from 8 to 28 per cent of the fed labeled n-alkanes are recovered in the cuticular lipids. Most of the radioactivity recovered from feeding the C₂₃ n-alkane and a significant amount from the C₂₅ was found as a secondary alcohol in the form of a wax ester. The C₂₉ and C₃₁ n-alkanes were recovered primarily unchanged as the n-alkane. Eighty-five per cent of injected acetate incorporated into the hydrocarbon fraction is in the branched hydrocarbons. These results show that the insect synthesizes its branched hydrocarbons, whereas a large part of the normal hydrocarbons can be dietary.     

Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly Sarcophaga bullata.

Ecdysterone has been shown to stimulate hydrocarbon biosynthesis in Sarcophaga bullata at pupariation. When post-feeding larva were treated with ³H-acetate 10·5 hr after hormone administration, a 1·3 times greater quantity of ³H-acetate was incorporated into hydrocarbon in the ecdysterone injected insects than controls. A similar experiment with a 24 hr delay of ³H-acetate administration following hormone treatment resulted in 3·3 times greater incorporation into hydrocarbon of treated animals.Isolated integuments synthesize hydrocarbon from acetate better than internal tissues, and the integuments of ecdysterone-treated insects incorporate acetate into hydrocarbon 9·8 times better than integuments of control insects. This indicates that cuticular hydrocarbon biosynthesis not only occurs in the integument, but that a locus of regulation is present in the integument.     

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.     

Effects of Petroleum Hydrocarbons on Plant Litter Microbiota in an Arctic Lake

The effects of petroleum hydrocarbons on the microbial community associated with decomposing Carex leaf litter colonized in Toolik Lake, Alaska, were examined. Microbial metabolic activity, measured as the rate of acetate incorporation into lipid, did not vary significantly from controls over a 12-h period after exposure of colonized Carex litter to 3.0 ml of Prudhoe Bay crude oil, diesel fuel, or toluene per liter. ATP levels of the microbiota became elevated within 2 h after the exposure of the litter to diesel fuel or toluene, but returned to control levels within 4 to 8 h. ATP levels of samples exposed to Prudhoe Bay crude oil did not vary from control levels. Mineralization of specifically labeled ¹⁴C-[lignin]-lignocellulose and ¹⁴C-[cellulose]-lignocellulose by Toolik Lake sediments, after the addition of 2% (vol/vol) Prudhoe Bay crude oil, motor oil, diesel fuel, gasoline, n-hexane, or toluene, was examined after 21 days of incubation at 10°C. Diesel fuel, motor oil, gasoline, and toluene inhibited ¹⁴C-[lignin]-lignocellulose mineralization by 58, 67, 67, and 86%, respectively. Hexane-treated samples displayed an increase in the rate of ¹⁴C-[lignin]-lignocellulose mineralization of 33%. ¹⁴C-[cellulose]-lignocellulose mineralization was inhibited by the addition of motor oil or toluene by 27 and 64%, respectively, whereas diesel fuel-treated samples showed a 17% increase in mineralization rate. Mineralization of the labeled lignin component of lignocellulose appeared to be more sensitive to hydrocarbon perturbations than was the labeled cellulose component.     

Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph

Lipophorin, radiolabelled in the protein or diacylglycerol moiety, was purified from adult locusts injected previously with [¹⁴C]protein hydrolysate or sodium[1-¹⁴C]palmitate. The radiolabelled lipophorin was injected into adult male locusts and haemolymph samples taken periodically to determine the rate of disappearance of radioactivity from the haemolymph. Lipophorin was also purified from locusts that had been injected four days previously with ([¹⁴C]protein)-lipophorin to demonstrate that the radioactivity observed in the haemolymph at this time is due to radiolabelled lipophorin. The results indicate that the half-life of the protein component of lipophorin in resting insects is about 5–6 days whereas that of the diacylglycerol component is only about 2–3 hr.The results are consistent with the hypothesis that lipophorin functions as a “reusable shuttle” to transport a variety of lipid classes between sites of absorption, storage and utilisation.     

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.     

Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH-I and 10, 11-epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH-III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [³H]JH-III and to a lesser extent JH-I. Other proteins from the hemolymph do not bind JH-III. Binding of JH-III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH-III. It is concluded that lipophorin is the JH-III-specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH-III-specific binding site is probably located on apolipophorin I.     

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga,Botryococcus braunii (Race A)

Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [¹⁴C]acetate. Incorporation of ¹⁴C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.     

Inhibition of hydrocarbon biosynthesis in the housefly by 2-Octadecynoate

The elongation of [9,10-³H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ²⁼) inhibited the in vivo incorporation of [1-¹⁴C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-¹⁴C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-³H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-¹⁴C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.     

Effects of light on terpene hydrocarbon synthesis in Pinus pinaster

The biosynthesis of terpene hydrocarbons has been investigated in maritime pine (Pinus pinaster Ait.) seedling primary leaves under light and darkness and with different precursors. Impossible in darkness, the synthesis of monoterpenes (mainly α- and β-pinene) is strongly activated by light. Only ¹⁴C-carbonate and ¹⁴C-acetate can be incorporated into monoterpenes. Activation by light is comparatively much more effective for seedling leaves previously cultivated under short days than in leaves from seedlings given long days. The spectral bands which are efficient for the synthesis of monoterpenes are located around 480 and 685 nm with ¹⁴C-carbonate and 480 and 630 nm with l-¹⁴C-acetate. Furthermore, this light activation does not occur if leaf pieces instead of whole leaves are used for the incorporation experiments. When 2-¹⁴C-mevalonic acid and 1-¹⁴C-isopentenyl pyrosphosphate are applied as precursors, no radioactivity is recorded in monoterpene hydrocarbons even after light exposures. In contrast, sesquiterpene hydrocarbons (caryophyllene and humulene) are easily synthesized under light or darkness in intact or fragmented leaves from the different precursors of photosynthetic or exogenous origin. From these results the compartmentalization in the synthesis of C₁₀ and C₁₅ hydrocarbons appears clear. There is a metabolic cooperation between the photosynthetic tissues and the specific site of elaboration of C₁₀ hydrocarbons, which site is located in the parts where the epithelial cells of resin ducts are functional. The synthesis of sesquiterpene hydrocarbons takes place in the whole leaf without activation by light.