Identification accumulation and biosynthesis of the cuticular hydrocarbons of the southern armyworm Spodoptera eridania cramer lepidopteraNoctuidae.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 16:19-30 (1991) Identification, Accumulation, and Biosynthesis of the Cuticular Hydrocarbons of the Southern Armyworm, Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) Lin Guo and Gary J. Blomquist Department of Biochemistry, Uninersity of Nevada, Reno, Nevada The accumulation and biosynthesis of cuticular and internal hydrocarbons in the Southern armyworm, Spodoptera eridania, were examined at closely timed intervals during larval and pupal development. Gas chromatography-massspectrometry (GC-MS) was used to identify n-alkanes, monomethylalkanes, and dimethylalkanes ranging in chain length from 23 to 35 carbons. The amount of cuticular hydrocarbon stayed relatively constant duringeach stadium, while the amount of internal hydrocarbon increased dramatically during the first half of each larval stadium, presumably to replace the cuticular hydrocarbon lost o n the shed cast skin with each molt. The accumulation of internal hydrocarbon was mirrored by large increases in the rate of incorporation of labeled acetate into the hydrocarbon fraction. Hydrocarbon production fell to very low rates during the latter part of the fourth and fifth larval stadia. Relatively high rates of hydrocarbon production were observed during the first and last one-third of the pupal stage and essentially all of the hydrocarbons produced during this stage remained internal. These data document large changes in the rates of hydrocarbon production during development in S. eridania and suggest that most of the hydrocarbon produced during each stage was stored internally and then transported to the cuticle of the next stage. Key words: hydrocarbon composition, hydrocarbon accumulation, hydrocarbon biosynthesis INTRODUCTION Hydrocarbons are important constituents of the cuticular lipids of insects, where they serve essential roles in preventing water loss and are important in many species in chemical communication [l-41. They have been studied exAcknowledgments: Supported in part by a grant (CAM 8900879) from the USDA-CRGO. We thank Murray Hackett for CC-MS analysis and David Quilici and Linda Mead for rearing the Southern armyworm colony. Received July26,1990; accepted August 24,1990. Address reprint requests to Lin Cuo, Department of Biochemistry, Howard Science Building, Universityof Nevada, Reno, N V 89557-0014. 0 1991 Wiley-Liss, Inc. 20 Cuo and Blomquist tensively in a number of insect groups and several recent reviews describe these studies [2-41. Cuticular hydrocarbons are often present as complex mixtures of straightchain, methyl-branched, and unsaturated components . The proportion of each class of hydrocarbon show large variations in different insect species . The hydrocarbon composition can be affected by temperature [ 5 ] ,habitat , and developmental stage . The relationship between low cuticular permeability and the occurrence of long-chain methylalkanes in cuticular hydrocarbon mixtures has been demonstrated in several insect species . Variations in hydrocarbon biosynthesis at different developmental stages were observed in several insects. Newly molted adult American cockroaches, Periplaneta arnericana, have relatively low levels of hydrocarbon biosynthesis while older ones have a higher rate of biosynthesis . Two periods of rapid accumulation of cuticular hydrocarbon were observed in the development of Snrcophagu bullata , the first occurring during pupation and the 3 day period following pupation, the second occurring during the 4 day period preceding the pupal-adult ecdysis. The biosynthesis of hydrocarbons was stimulated by ecdysterone in S. bullata [lo]. Hydrocarbons were synthesized at specific time periods during larval and pupal development in the cabbage looper, Trichoplusia ni. The rate of hydrocarbon biosynthesis increased dramatically after each molt [7,11] during the feeding stages and fell to very low levels during the wandering stages. Most insects are susceptible to dessication at certain times during development . Thus, it was of interest to determine the identity of the cuticular and internal hydrocarbons and to examine their accumulation and rates of formation in the Southern armyworm. These studies are reported herein. MATERIALS AND METHODS Insects Southern armyworms were reared according to the procedure reported in detail elsewhere [ 131. Regimes for rearing insects were photoperiod, 14:lO (L:D); temperature, 25°C k 1”C:22”C & 1°C(L:D); and relative humidity, ca 50-60%. The larval diet was essentially the same as described by Rehr et al. , but contained foliage of lima bean, rather than red kidney bean. Radioactive Substrate Sodium [ l-I4C]acetate (45 mCi/mmol) was obtained from ICN Biomedicals Inc., Costa Mesa, CA. In Vivo Studies Larvae of known ages were injected with 1.0 pl each of [1-14C]acetate(0.5 pCUp.1) just under the last pair of prolegs. Pupae of known ages were injected through the last two abdominal segments. A microcapillary injection technique, as described in Dwyer et al. [ll], was used to minimize trauma to the insect. Following a 2 h incubation at about 27T, insects were killed by immersion in hexane. Three groups of five insects each were used for each time point. Values reported represent the mean ? standard deviation. HydrocarbonProduction in the Southern Armyworm 21 Extraction, Separation, and Identification Cuticular lipids were extracted by rinsing insects in redistilled hexane (5 ml per group of five insects) for 5 min, removing the solvent, and adding 2 ml of hexane for 1 min. The extracts were combined and reduced in volume under a stream of N2. Hydrocarbons were isolated by chromatography on minicolumns (6 x 0.5 cm) of Biosil-A (Bio-RadLabs, Richmond, CA)eluted with 8 ml of hexane. Internal lipids were extracted by the method of Bligh and Dyer  and the various lipid classes were separated by thin-layer chromatography (Silica gel type H). The plates were developed in hexane-diethyl ether (95:5)and visualized under UV light after spraying with Rhodamine 6G (0.1% in methanol). The hydrocarbons were extracted from the silica gel with diethyl ether. A known amount of n-Octacosane was added as an internal standard at the time of extraction. n-octacosane was not present in S. eriduniu hydrocarbon at above 0.5% level of the total hydrocarbon at any time during the stages studied. All gas chromatographic analyses were performed on a Hewlett Packard 5890 gas chromatograph which was controlled by a Hewlett-Packard 3393A integrator GC* terminal. Samples were analyzed on a 30 m x 0.32 mm I.D, 0.5 pm stationary phase DB5 column, The oven was temperature-programmed from 200 to 310°C at 5"C/min, then held at 310°C for 15 min. The carrier gas was helium, at a flow rate of 2.5 ml/min, and the carrier gas plus make-up gas at a total flow rate of 30 ml/min. Gas chromatography-massspectrometry (GC-MS) analyses were performed on a Finnigan 4023 mass spectrometer interfaced with an INCOS data system. The GC-MS system operated at 70 eV (GC condition: 30 m x 0.32 mm DB5 column, 30-200°C at 20"C/min, 200-28O0C/min at 5"C/min, held at 280°C for 15 min). The carrier gas was helium at 8 PSI head pressure, linear velocity about 30 cm/s. All GC peak retention times were expressed as equivalent chain lengths (ECLs). ECL for each hydrocarbon was calculated by comparing the retention time of a given peak with known n-alkanes. Mass spectra of methylalkanes were interpreted according to the criteria of Nelson  andBlomquist et al. . Determination of Radioactivity Samples were assayed for radioactivity on a Beckman LS 1701liquid scintillation counter in a 10 ml fluor solution containing 0.4% 2,5-diphenyloxazole in toluene at an efficiency of ca 96% for carbon-14. RESULTS Hydrocarbon Structure and Composition The major components of the cuticular hydrocarbons of the Southern armyworm larvae are listed in Table 1. The same major components are present on larvae, pupae, and adults, with differences noted in the percentage of each component. The ECL values indicated that methyl-branched hydrocarbons are the predominant components with the remainder being n-alkanes, principally n-C27, n-C29, and n-C31 (Table 1).No unsaturated hydrocarbons were found. The percentages of the methyl-branched hydrocarbons increased *Abbreviations used: ECL = equivalent chain length; FAS = fatty acid synthesis; GC = gas chromatography, MS = mass spectrometry. ECLb 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 29.27 29.37 30.00 31.00 31.26 31.37 Component n-Nonadecane n-Eicosane n-Heneicosane n-Docosane n-Tricosane n-Tetracosane n-Pentacosane n-Hexacosane n-Heptacosane n-Octacosane n-Nonacosane 13-Methylnonacosane 7-Methylnonacosane n-Triacontane n-Hentriacontane 9-, 11-, 13-Methylhentriacontane 7-Methylhentriacontane Peak” 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1.5 1.4 1.4 0.9 1.0 0.7 0.8 0.8 2.7 1.8 16.2 2.1 0.6 1.9 6.5 1.3 2.2 3rd Instar 2.8 0.4 0.7 0.2 0.9 0.4 0.6 0.7 0.7 2.1 1.7 15.3 1.6 0.6 1.8 5.1 1.2 1.8 1.5 1.4 1.3 1.0 0.7 0.6 0.7 0.7 3.2 1.6 11.0 2.3 0.7 1.4 2.9 1.3 3.0 0.4 0.5 0.3 0.8 0.3 0.2 2.6 0.7 5.5 0.7 3.9 1.5 0.4 0.5 1.7 3.5 Percentage composition‘ 4th 5th Instar Instar Pupae 0.8 1.4 1.0 0.5 0.5 0.3 0.3 1.1 0.5 10.8 0.9 12.9 0.2 0.2 0.5 1.5 0.7 Adult (continued) 140,337,450 168,309,450 196,281,450 112,344,450 434 268 282 296 310 324 338 350 364 380 394 408 196,253,422 112,337,422 420 Diagnostic mass spectral ion fragment TABLE 1. Composition of the Cutidar Hydrocarbons of the Southern Armyworm S. eridattia Third, Fourth, and Fifth h t a r Larvae; Pupae and Adults* 35.67 11,23-Dimethylpentatriacontane 28 2.1 39.6 60.4 2.3 32.5 67.5 12.8 0.4 1.2 2.3 2.3 23.2 4.5 25.4 74.6 14.0 13.8 2.8 29.5 70.5 2.6 1.8 2.8 6.2 17.0 0.6 1.2 2.6 2.1 25.3 5.1 34.6 65.4 29.8 0.7 2.4 3.4 6.8 6.5 0.9 1.1 0.7 0.9 7.8 2.5 3.7 1.7 2.3 14.6 0.4 1.3 1.3 a.7 14.I 0.6 1.4 1.2 0.9 14.7 Adult Percentage composition‘ 4th 5th Instar Instar Pupae 168,351,490 168,365,504; 196,337,504 168,196, 351,379,518 462 140,365,476; 168,337,476; 196,309,476 168,196,323, 351,490 476 450 Diagnostic mass spectral ion fragment *Theinsects used were 24 hr-oId after the molt to the stage indicated. “Peak number refer to peaks identified in Figure 1. bECL = equivalent chain length. ‘Number represent the average of three determinations, 10 insects per group. The range of values were less than 20% of the mean. 35.75 10.8 34.00 34.15 34.32 35.31 n-Tetratnacontane Unknown 11-Methyltetratriacontane 11-, 13-Methylpentatriacontane 24 25 26 27 29 Unknown Total straight chain hydrocarbons Total methyl-branched hydrocarbons 0.2 1.1 1.9 2.5 33.61 11,21-Dimethyltritriacontane 23 18.8 32.00 32.35 32.65 33.00 33.32 n-Dotriacontane Unknown Unknown n-Tritriacontane 9-, 11-, 13-Methyltritriacontne 18 19 20 21 22 0.6 1.7 1.0 1.3 14.4 ECLb Component 3rd Instar Peak’ TABLE 1. (Continued) 24 Cuo and Blomquist steadily from third (60.4%) instar larva to fifth instar larva (70.5%),and with the pupa containing the highest percentage of methyl-branched hydrocarbon (74.6%)(Table 1). GC-MS was used to characterize each of the major components. The mass spectra of GC peaks (Fig. 1)22 and 23 are presented in Figure 2A and B and they are representative of the spectra obtained. Under electron impact, methylalkanes fragment preferentially on either side of a methyl branch. The mass spectrum of peak 22 (Fig. l),which has the ECL 33.32, is identified as a mixture of 9-, 11-and 13-methyltritriacontane (Fig. 2A). The major isomer appears to be the 11-methyl component which gives rise to ions at miz 1681169 and 336/337. The strong m/z 168/169 and 336/337 ions are interpreted as arising from a-cleavage on either side of the methyl-branch point. The ions at m/z 168 and 336 result from the loss of a hydrogen atom from the fragment at 169 and 337, respectively. The preferred a-cleavage is the one which loses the bigger radical, the 22-carbon tail. Isomers with methyl groups at positions 9 and 13 are also present. Peak 23 from Figure 1is interpreted as 11,21-dimethyltritriacontane(Fig. 2B). The two clusters with a significant even-mass ion at m/z 1681169 and 1961197 are interpreted as arising from the a-cleavage internal to the methyl-branch points which yield a 12-carbon and a 14-carbon fragment ion, respectively. The two ion clusters with only a significant odd-mass ion at m/z 351 and 379 are interpreted as arising from the a-cleavage external to the methyl-branch points yielding a 25-carbon and a 27-carbon fragment ion, both of which have a methyl branch which suppresses the formation of the even-mass ions . The n-alkanes were identified by GC retention times and their characteristic mass spectra. The n-alkanes have carbon numbers from C19 to C34 with odd numbered components predominating, mainly C27, C29, and C31 (Table 1). The mass spectra of the cuticular hydrocarbon from fourth instar larvae and pupae were also obtained and the same hydrocarbon components were present as in fifth instar larvae. Accumulation of Hydrocarbon Essentially all of the cuticular hydrocarbons on fourth instar larvae just prior to ecdysis were recovered on the cast skins (Table 2). Of the 11.0 2 1.2 pg of internal hydrocarbon in the fourth instar larvae, 3.7 ? 0.7 pg were found in the cuticular hydrocarbons of newly emerged fifth instar larvae and 7.1 ? 0.8 were recovered in internal extracts (Table 2). These data strongly suggest that Fig. 1. CC trace of the cuticular hydrocarbonsof the Southern armyworm, S. eridania 1-day-old fifth instar larvae. The nomenclature used for GC peaks is described in Table 1. 25 Hydrocarbon Production in the Southern Armyworm A I 1111 I 168 ,111 L 100 100 150 200 250 - - 50 -- - 337 309 11. 365 It II 111 I. - - "I 96 1111 I II....,, I II .,,I . I ,I . .I1 1 ,, I Ill11 L 100- - - I I 351 196 - 50 -- I 323 -. ,I. .#I. I I ' ' 11, 351 ' , ' I, l ' ' ' ' l ' r ' ~ Fig. 2, Mass spectra of peak 22, Figure 1, identified as 9-, 11-, and 13-methyltritriacontane (A) and peak 23, Figure 1 , identified as 11,21-dirnethyltritriacontane(B). S. eridania loses all of its cuticular hydrocarbons during each molt and the internal hydrocarbon pool is used to provide hydrocarbons for the newly molted insect. The amount of cuticular hydrocarbon stayed relatively constant during each stadium while the amount of internal hydrocarbon increased dramatically during the first half of each stadium (Fig. 3 ) . During the fourth larval stadium, both the amount of cuticular and internal hydrocarbons increased l Guo and Blomquist 26 TABLE 2. Amounts of the Hydrocarbon Present in S. eridunia During the Fourth to Fifth Larval Ecdysis Hydrocarbon mass (Fglinsect 2 SD) Cuticular Internal Total Stage 48 h after 3rd to 4th stadium ecdysis Shed cast skin Immediately following fourth to fifth stadium ecdysis - - 70 60 5.4 4 0.6 5.5 ? 0.6 -3.7 % 0.7 - 11.0 + 1.2 16.42 1.8 7.1 -t 0.8 10.8 k 1.5 - Cuticular hydrocarbons Internal hydrocarbons Fig. 3. Amounts of cuticular and internal hydrocarbons during fourth and fifth instar larval and pupal development. E, ecdysis. during the first 16 h, and then the amount of cuticular hydrocarbons remained almost constant while the amount of internal hydrocarbon increased until the next ecdysis. During the fifth stadium, the amount of cuticular hydrocarbons remained essentially constant whereas the accumulation of internal hydrocarbon increased dramatically following the fourth to fifth stadium molt, and then declined about 40 h later. During the pupal stage, the amount of cuticular hydrocarbon remained relatively constant, but the amount of internal hydrocarbon increased up to 24 h, leveled off until 96 h, and then declined significantly (Fig. 3). Biosynthesis of Hydrocarbon The incorporation of [l-I4C]acetateinto hydrocarbons was used to estimate the rate of h drocarbon formation. Large fluctuations in the percent incorporation of [I-'Clacetate into cuticular and internal hydrocarbons during fifth stadium larval and pupal development were observed (Fig. 4). Consistent with the data obtained from studies measuring hydrocarbon accumulation, a high rate of incorporation of [1-l4C]acetateinto cuticular and internal hydrocarbons Hydrocarbon Production in the Southern Armyworm 1.2 - 1.0 - = Cuticular hydrocarbons Internal hydrocarbons 27 T T I hours 0.0 E 48 E 4th I 48 96 - 48 96 5th LARVA PUPA 144 192 - Fig. 4. Incorporation of [1-’4C]acetate into cuticular and internal hydrocarbons during larval and pupal development. E, ecdysis. following the fourth to fifth instar molt was observed. Unlike the decline in the amount of internal hydrocarbon of fifth instar larvae at about 40 h after fourth to fifth stadium molt, the rate of incorporation of [l-14C]acetate into internal hydrocarbon declined much earlier. The fifth instar larvae showed the highest rate of hydrocarbon production at about 16 h after the fourth to fifth stadium molt (Fig. 4). Whereas almost no incorporation of [l-14C]acetateinto cuticular hydrocarbons was observed during pupal development, a dramatic increase of [l-I4C]acetate incorporation into internal hydrocarbon was observed irnmediately after pupal ecdysis (Fig. 4). This decreased from 48-96 h after pu a tion. After 96 h, there was again an increase in the incorporation of [l-lC] acetate into internal hydrocarbons which remained high until the end of the pupal stage. Other than at time points immediately following ecdysis and at 16 h after pupation, no labeled hydrocarbon was recovered in cuticular extracts from pupae. f- DISCUSSION The cuticular lipids of most insect species contain an isomeric mixture of methyl-branched alkanes [2-41 which commonly include mono- and dimethylalkanes and less commonly tri- and tetramethylalkanes [16-181. The cuticular n-alkanes of most insect species occur as continuous homologous series, and n-C23, n-C25, n-C27, nC-29, and n-C31 usually predominate .The cuticular hydrocarbons of the Southern armyworm, S. eridania, contain n-alkanes and both mono- and dimethyl-branched alkanes and have a high proportion 28 Cuo and Blomquist of methyl-branched alkanes. The methyl-branched alkanes of S. eridaniu were similar in structure to those found in many other insects [l-41, including different families of insects. However, some common hydrocarbon components in insects, such as 2-methyl and 3-methylalkanes and alkenes were not found in S. eridaniu. The amount of methyl-branched hydrocarbons vary considerably among different insect species. The methyl-branched hydrocarbons range from 98% of the cuticular hydrocarbons in 7'. ni  to 2% in Eurychora sp. "1. This may be related to their ability to prevent water loss. In a comparison of three species of tiger beetles, Cicindela obsoletu, C. oregonu, and C. tranquebaricu, Hadley and Schultz  found that the species with the most hydrocarbon of the highest saturation and methyl-branching had the least water loss. S. eridurtiu larval cuticular hydrocarbons were comprised exclusively of saturated hydrocarbons with 60-70% of them being methyl-branched components. The percentage increase of the methyl-branched hydrocarbon from third instar larva to fifth instar larva may suggest that as the insect size increases, the prevention of water loss becomes more crucial. The pupal stage contains the highest percentage of methyl-branched hydrocarbon and may reflect the fact that the pupal stage is most sensitive to desiccation. All of the cuticular hydrocarbon on the surface of S. eriduniu is apparently lost on the cast skin during the fourth to fifth instar molt. About one-third of the internal hydrocarbons of S. eridania just prior to ecdysis were present on the cuticle of the newly emerged insect and about two-thirds of them remained internal. In a study with the cabbage looper, T. ni, it was shown that the cabbage looper doesn't reabsorb its cuticular hydrocarbons prior to molting. Instead, in the three different types of molts examined (larva-larva [ll]; larva-pupa and pupa-adult ) they are discarded with the cast skins. The larva-larva molt of S. eridunia also showed that no cuticular hydrocarbons were reabsorbed and suggested that this may be a common phenomena in insects. Hydrocarbons are synthesized by the cells associated with the epidermis, probably the oenocytes [3-41 and are subsequently transferred to an undetermined internal storage site. In several insect species, including the American cockroach and migratory locust [21,22], a major lipid class associated with lipophorin is hydrocarbon, and the hydrocarbons associated with the lipophorin have the identical components as do the cuticular hydrocarbons. Thus, lipophorin is suggested as a storage site for hydrocarbons. At present, the exact role of lipophorin in the storage and transportation of cuticular hydrocarbon is not clear. In S. eridania, the large fluctuation in the accumulation and synthesis of hydrocarbons observed during the last larval and pupal stage clearly indicated the existence of a regulatory mechanism for the production and deposition of hydrocarbon on the cuticle. In both the last larval and pupal stage, the decline in the rate of incorporation of [l-14C]acetateinto internal hydrocarbon occurs much earlier than does the decrease in internal hydrocarbon accumulation. Thus as the rate of hydrocarbon synthesis decreases, the insects accumulate more hydrocarbon internally. From studies with T. ni, de Renobales et al.  suggested the existence of two pools of internal hydrocarbon, one destined for the cuticle (synthesized some time before)and another one that remains internal at all times. The study reported here on S. eridania is consistent with this suggestion. The variation Hydrocarbon Productionin the Southern Armyworm 29 in accumulation during fifth instar larval and pupal stadium may only reflect the change in one of the internal hydrocarbon pools. In several other insect species studied [7-9, 111, the developmental regulation of hydrocarbon biosynthesis and accumulation was also observed. The relationship between the regulation of hydrocarbon synthesis and the regulation of fatty acid synthesis in insects needs to be further studied. In T. ni, during the fifth larval instar, FAS activity correlated with the synthesis of hydrocarbons and other lipid classes. However, throughout the pupal stage, negligible level of FAS activity was found in spite of the very high rate of hydrocarbon formation . This suggested that the biosynthesis of long-chain methyl-branched hydrocarbons is by a different system than that of fatty acids in T. ni. Further work with S. eridaniu is necessary to understand the enzymology, regulation, and transport of hydrocarbons. LITERATURE CITED 1. Blomquist GJ, Jackson LL: Chemistry and biochemistry of insect waxes. Prog Lipid Res 17,319 (1979). 2. Blomquist GJ, Dillwith JW: Cuticular lipids. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol 3, pp 117-154 (1985). 3. Blomquist GJ, Nelson DR, de Renobales M: Chemistry, biochemistry, and physiology of insect cuticular lipids. Arch Insect Biochem Physiol6,227 (1987). 4. Lockey KH: Lipids of the insect cuticle: Origin, composition and function. Comp Biochem Physiol89B, 595 (1988). 5. Hadley NF: Epicuticular lipids of the desert tenebrionid beetle, Eleodes urmata: seasonal and acclimatory effects on composition. Insect Biochem 7,277 (1977). 6. Armold MT, Blomquist GJ, Jackson LL: Cuticular lipids of insects - 111. 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