The compositoin of cuticular hydrocarbons of the cereal aphids Sitobion avenae F. (Homoptera, Aphididae)

Comp. Biochem. Physiol. Vol. 94B, No. 4, pp. 723-727, 1989

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THE COMPOSITION OF CUTICULAR HYDROCARBONS OF THE CEREAL APHIDS SITOBION A VENAE F. (HOMOPTERA, APHIDIDAE) ESTERAHEBANOWSKA,*EDMUND MALII~SKI,*JAN NAWROT,t MARIARUSZKOWSKA,~" KALEVIPII-ILAJA~ and JANUSZSZAFRANEK* *Institute of Chemistry, University of Gdafisk, 80-952Gdafisk, Sobieskiego 18, Poland; tInstitute of Plant Protection, Miczurina 20, 60-318 Poznafi Poland; and :~Departmentof Chemistry, University of Turku, SF-20 500 Turku, Finland (Received 13 April 1989)

Abstract--1. The cuticular hydrocarbons of the cereal aphids, Sitobion avenae F. were studied by capillary column chromatography and mass spectrometry. 2. n-Alkanes, 2-, 3-, 4-, 5-, 6-, 10-, 11-, 12- and 13-monomethylalkanes, 7,11-, 11,15-, 13,17- and 5,11-dimethylalkanes were found in cuticular lipids. 3. The results obtained are significantlydifferent from these of pea aphid, where only n-alkanes were found. 4. The n-alkanes of cereal aphids range from 23 to 35 carbon atoms with the predominance of odd over even members. 5. There are terminally branched hydrocarbons 2-methyl and 3-methyl-alkanes rarely found together in cuticular lipids of insects.

INTRODUCTION Hydrocarbons usually constitute a major group of compounds of the insect cuticular lipids (Lockey, 1980, 1985, 1988). Among them not all come from insect diet. There is biochemical evidence that insects synthesize most of their cuticular hydrocarbons (Nelson, 1978). Therefore, hydrocarbon composition, is an expression of genotype and as such, it is available as a taxonomic indicator. For example, the chemotaxonomic values of hydrocarbon composition has been described by Lockey (Lockey, 1978a,b, 1984) in family Tenebronidae and Baker et al. (Baker, 1984) in genus Sitophilus. Knowledge of composition of cuticular lipids is also important due to their physiological functions: (1) preventing desiccation of the insects, (2) preventing penetration by microorganisms and toxic substances, (3) serving as chemical messengers or short distance pheromones as well (Lockey, 1988). Cereal aphid, Sitobion avenae F., is a dangerous pest of cereals which is of great importance in the countries with intensive agriculture production. In Europe cereal aphids appear every year on all cereal species mainly on the ears. Thus these are known as the pest of generative parts of the plants. They appear on the plants most frequently just after the earing of cereals. In the present work the cuticular hydrocarbon mixture of adult Sitobion avenae has been analysed by gas chromatography (GC) and combined gas chromatography-mass spectrometry (GC-MS). MATERIALS AND METHODS Cereal aphids were collected from ears of the rye in the field during the abundant appearance of the species during

milk mature of grains. The material obtained was heterogenous (600 aphids in different stages of development, 120 adults (female viviparae) and 480 larvae). Mixture of cuticular lipids was obtained by immersing insects in 200 cm3 methylene chloride and leaving them in the solvent for 2 weeks. The extract was filtered and the solvent was removed in a rotatory evaporator at reduced pressure. Hydrocarbons were separated from other lipids by elution with hexane (100 cm 3) on a column packed with silica gel (Merk) activated at 150°C for 24hr. Separation of hydrocarbons was achieved by gas chromatography using a 40 m glass capillary column coated with OV-1 and SE-52 liquid phases (column internal diameter 0.3 mm, film thickness 0.12 #m). GC analyses were carried out using a Varian Aerograph 1400 modified to accept a capillary column. Helium was used as a carrier gas. The Kovhts retention indexes were determined at 240, 260 or 280°C. The k' values were greater than 5. GC-MS measurements were carried out with VG Micromass 7070 E mass spectrometer coupled with a Dani 3800 gas chromatograph. A fused silica capillary column with SE-30 liquid phase was used. The mass spectra were recorded at 70 eV. RESULTS

Hydrocarbon fraction constitutes a low percentage of the total extracted lipids. The gas chromatogram of the hydrocarbon mixture is given in Fig. 1. The contents of individual hydrocarbons obtained by GC peaks integration and the identification obtained by GC-MS and by Kov~its retention indexes are given in Table 1. The hydrocarbon fraction of Sitobion avenae contains n-alkanes, terminally branched monomethylalkanes (iso- and anteiso-), internally branched monomethylalkanes and dimethylalkanes.

723

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ESTERA HEBANOWSKAet al. 47

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n-Alkanes (47.4% of hydrocarbon fraction) form a homologous series ranging from C23 to C3s with an odd (37.1%) to even (10.3%) predominance. They were identified by coinjection with standards and confirmed by GC-MS measurements, too. n-C25 and n-C27 hydrocarbons are the most abundant in this series. There are 43.4% monomethylalkanes in hydrocarbon fraction. Monomethylalkanes consist of 21.6% terminally branched (2- and 3-methyl) isomers and 21.8% internally branched monomethylalkanes (4-methyl to 13-methyl). 2-Methylalkanes (13.9% of hydrocarbon fraction) form a homologous series ranging from C24 to C30. They were identified by Kov~.ts retention indexes (Ku~mierz et aL, 1985) and by GC-MS measurements. The characteristic ions for 2-methylalkanes at (M-43) + and (M-15) + were observed. There are three 3-methylalkanes (7.7% of hydrocarbon mixture) with even carbon numbers (C24, C26 and C2s). The identification was performed by means of the Kov~its retention indexes and confirmed by GC-MS. The characteristic ions for these compounds at (M-29) ÷ were observed. There is one 4-methyl alkane, viz. 4-methylhexascosane. This hydrocarbon was identified both by retention index and G-C-MS where the characteristic ion for 4-methylalkane (M-43) + was observed (Fig. 2). In the same

way, 5-methylalkanes (2.8% of total hydrocarbon mixture) with even carbon numbers (C26, C2s), were identified. The mass spectra of these methylalkanes contain characteristic ions at m/z (m-57) ÷. There is one 6-methylisomer (C27). This alkane was identified by Kovhts retention index and confirmed by GC-MS. There are two pairs of characteristic ions for 6methylhexacosane at m/z 98/99 and m/z 308/309 (Fig. 3). There is one 8-methyl isomer (C27) in hydrocarbon fraction as well. This alkane was identified both by retention indexes and GC-MS (two pairs of characteristic ions at m/z 126/127 and m/z 280/281). The other monomethylalkanes (10-, 11-, 12-, 13-methyl: 16.2% of hydrocarbon mixture) as the more internally branched isomers are not separated so well. They were only identified by GC-MS. The chromatographic peaks labelled as 29, 34, 39, 40, 41 and 45 were identified as dimethylalkanes (5.8% of hydrocarbon mixture). All these dimethylalkanes have odd carbon numbers (C31, C33 and C3s). The branching points were established by GC-MS. The mass spectra of chromatographic peak 29 (Fig. 4) shows the fragmentation pattern of 7,11-dimethylnonacosane which is consistent with the ion pairs at m/z 182/183 (Cl3) and m/z 280/281 (C20), and enhanced fragment ions at m/z 112/113 (C8) and m/z 350/351 (C25).

Aphid cuticular hydrocarbons

725

Table 1. Composition of hydrocarbons of the cereal aphid, Sitobion avenae F. GC peak no.

Kovhts retention indexes

Intensity (%)

n-tficosane 2-methyltficosane

2300 2363

0.7 0.1

3-methyltricosane n-tetracosane 2-methyltetracosane

2375 2400 2465

0.2 1.1 2.8

n-pantacosane 7-methylpentacosane 5-methylpentacosane 2-methylpentacosane

2500 2543 2551 2564

11.4 0.3 1.0 3.4

10 I1 12 13 14 15 16

3-methylpentacosane n-hexacosane ll-methylhexacosane 8-methylhexacosane 6-methylhexacosane 4-methylhexacosane 2-methylhexacosane

2574 2600 2635 2639 2647 2661 2664

5.4 5.5 0.3 0.3 0.4 1.1 4.6

17 18

n-heptacosane ll-,13-methylheptacosane

2700 2736

12.1 1.4

19 20 21

7-methylheptacosane 5-methylheptacosane 2-methylheptacosane

2743 2753 2764

0.7 1.8 0.7

22 23 24

3-methylheptacosane n-octacosane 10-,12-methyloctacosane

2774 2800 2836

2.1 2.1 0.7

25 26 27 28

2-methyloctacosane n-nonacosane 1 l-methylnonacosane 2-methylnonacosane

2864 2900 2935 2963

0.8 8.0 5.7 1.5

29

7,1 l-dimethylnonacosane

2974

0.7

30 31 32 33

n-tfiacontane 12-methyltriacontane n-hentfiacontane ll-,13-methylhentfiaeontane

3000 3034 3100 3133

1.1 1.7 2.9 2.9

34

1I, 15-dimethylhentriacontane

3160

2.4

35 36

n-dotriacontane 10-,12-methyldotriacontane

3200 3234

0.5 1.0

37 38

n-tritriacontane 11-,13-methyltritriacontane

3300 3333

1.1 1.6

39

11,15- and 13,17-dimethyltritriacontane

3359

1.4

40

7,11-dimethyltritriacontane

3371

0.4

41

5,11-dimethyltritriacontane

3382

0.3

42 43 44 45

12-methyltetracontane n-pentatriacontane 13-methylpentatriacontane 13,17-dimethylpentatriacontane

3431 3500

0.7 0.2 0.9 0.6

C.B.P. 94/41~-H

Identification

Mass spectral data 324 (M) + 338(M) ÷, 323(M-15)+, 295(M-43) ÷ 309(M-29) ÷ 338(M) + 352(M~), 337(M-15)+, 309(M-43) ÷ 352(M~) 112/113, 280/281 84/85, 308/309 366(~), 351(M-15)÷ 323(M-43) + 337(M-29) ÷ 366(M~) 168/169, 238/239 126/127, 280/281 98/99, 308/309 70/71 336/337 380(M?), 365(M-15)+, 337(M-43) ÷ 380(M?) 168/169, 252/253; 196/197, 224/225 112/113, 308/309 84/85, 336/337 394(M?), 379(M-15)+ 351(M-43) ÷ 365(M-29) + 394(M?) 154/155, 280/281; 252/253, 182/183 393(M-15) +, 365(M-43)÷ 408(M~) 168/169, 280/281 422(M~),407(M-15) +, 379(M-43) ÷ 182/183, 280/281; 112/113, 350/351 422(M?) 182/183, 280/281 436(M~) 168/169, 308/309; 196/197, 280/281 168/169, 322/333; 210/211,252/253 450(M~) 154/155, 336/337; 182/183, 308/309 464(M~) 168/169, 336/337; 196/197, 308/309 168/169, 350/351; 280/281, 238/239 196/197, 322/333; 252/253, 266/267 182/183, 336/337; 112/113,406/407 182/183, 336/336; 84/85, 434/435 182/183, 336/337 493(M~) 196/197, 336/337 196/197, 350/351; 266/267, 280/281

Total no. of C atoms 23 24 24 24 25 25 26 26 26 26 26 27 27 27 27 27 27 28 28 28 28 28 28 29 29 29 30 30 31 30 31 32 33 32 33 33 34

35 35 35 35 35 36 37

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Aphid cuticular hydrocarbons

727

DISCUSSION

REFERENCES

The cuticular hydrocarbon composition of cereal aphid is relatively simple. The following hydrocarbons were detected: n-alkanes (C23 to C35), terminally branched monomethylakanes (2- and 3-methyl), internally branched monomethylalkanes (4- and 13methyl) and dimethylalkanes. Separation techniques employed were sufficient to separate the components of the mixture and chromatographic peaks presented individual compounds except internally branched monomethylalkanes. The mass spectra allow us to identify all components including not resolved internally branched monomethylalkanes. Dimethylalkanes are minor components of the cuticular hydrocarbon mixture. Their branching points are isoprenoidally spaced (73 I-, 11,15-, 13,17dimethylalkanes). These spacings are the commonly reported types of branched hydrocarbon (Lockey, 1988). Stdnsk~ et al. (1973) investigated cuticular waxes of pea aphid, Acyrthosiphon pisum, which belongs to the same family, Aphididae. Unfortunately, their analytical technique were insufficient to identify the minor components. Nevertheless, there are similarities in n-alkanes distribution profiles for both insects. Closer correlation between the branched hydrocarbon fractions could not be made. An unusual feature of cereal aphid is the presence of 2-methyl- and 3-methyl alkanes, which are rarely found together in cuticular lipids of insects (Lockey, 1988).

Baker J. E., Woo S. M., Nelson D. R. and Fatland C. L. (I 984) Olefines as major components of epicuticular lipids of three Sitophilus weevils. Comp. Biochem. Physiol. 77B, 877-884. Ku~mierz J., Malifiski E., Czerwiec W. and Szafranek J. 0985) Kov~its retention indices of high molecular weight monomethyl-, cyclopentyl-, cyclohexyl- and phenylalkanes. J. Chromatogr. 331, 219-228. Lockey K. H. (1978a) The adult cuticular hydrocarbons of Tenebrio molitor L. and Tenebrio obscurus F. (Coleoptera: Tenebronidae). Insect. Biochem. 8, 237-250. Lockcy K. H. (1978b) Hydrocarbons of adult Tribolium castaneum Hbst. and Tribolium confusum Duv. (Coleoptera: Tenebronidae) Comp. Biochem. Physiol. 61B, 401-407. Lockey K. H. (1980) Insect cuticular hydrocarbons. Comp. Biochem. Physiol. 65B, 457-462. Lockey K. H. (1984) Hydrocarbons of Metriopus depressus (Haag) and Renatiella scrobipennis (Haag) (Coleoptera: Tenebronidae) Insect. Biochem. 14, 65-75. Lockey K. H. (1985) Insect cuticular lipids. Comp. Biochem. Physiol. 81B, 263-273. Lockey K. H. (1988) Lipids of the insect cuticle: origin, composition and function. Comp. Biochem. Physiol. 89B, 595~45. Nelson D. R. (1978) Long chain methyl-branched hydrocarbons: Occurrence, biosynthesis and function. Adv. Insect. Physiol. 13, 1-33. S~nsk~, K., Ubik K., Holman J. and Streibl M. (1973) Chemical composition of compounds produced by the pea aphid, Acyrthosiphon pisum (Harris). Pentane extract of surface lipids. Coll. Czech. chem. Commun. 38, 770-780.