Cuticular methylalkanes of adult cockroaches, Blatta orientalis and Periplaneta americana

Comp. Biochem. Physiol. Vol. 85B, No. 3, pp. 567-572, 1986 Printed in Great Britain

0305-0491/86 $3.00 +0.00 Pergamon Journals Ltd

CUTICULAR METHYLALKANES OF ADULT COCKROACHES, B L A T T A O R I E N T A L I S AND P E R I P L A N E T A A M E R I C A N A K. H. LOCKEY and B. DULARAY Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK (Tel: (041)33~8855) (Received 4 February 1986)

Abstract--1. The cuticular methylalkanes of Blatta orientalis and Periplaneta americana have been analysed by gas chromatography and by combined gas chromatography-mass spectrometry and comprise homologous series of 3-methylalkanes (C25 to C28), internally branched monomethylalkanes (C25 to C29) and dimethylalkanes (C25, C27 & C29). 2. The methylalkanes of B. orientalis and P. americana are compared with those of other cockroach species.

INTRODUCTION

Methylalkanes were identified from their mass spectra which were obtained with a 16F V.G. Micromass gas chromatograph-mass spectrometer, using a capillary column similar to the one used in GC analyses. The methylalkanes of both species were temperature programmed from 100-200°C at 10°C/min and then from 200-300°C at l°C/min. The temperature of the ion source was 230°C and the ionization voltage, 70eV. Helium at a flow rate of 2 ml/min was used as the carrier gas. The mass spectrometer was interfaced to a V.G. Data System 2000 and mass spectral scans (MS scans), ranging from m/z 20-m/z 650, were taken repetitively at a cycle time of 3.5 see. MS scans were selected for examination from the Total Ion Count (TIC) chromatogram of each mixture. Background was subtracted by the data system before the MS scans were examined. The mass spectra of methylalkanes are interpreted according to the criteria proposed by McCarthy et al. (1968), Nelson et al. (1972), Nelson (1978) and Pomonis et al. (1978, 1980).

While investigating the hydrocarbons extracted from cuticular grafts of Blatta orientalis and Periplaneta americana, as part of a wider investigation into donor-recipient interactions of insect cuticular grafts (Lackie, 1983), it became clear that previous analyses of the cuticular hydrocarbons of B. orientalis (Jackson and Baker, 1970; Tartivita and Jackson, 1970) and P. americana (Baker et al., 1963; Beatty and Gilby, 1969; Gilby and Cox, 1963; Jackson, 1972) had overlooked the homologous series of methylalkanes of the two species. The present work examines the cuticular methylalkanes of B. orientalis and P. americana by gas chromatography (GC) and by combined gas chrom a t o g r a p h y - m a s s spectrometry ( G C - M S ) . MATERIALS AND METHODS Male and female adults of both species were killed by freezing at - 20°C and then washed in re-distilled petroleum spirit (boiling range 60-70°C) at room temperature for 5 min to extract cuticular lipid. Details of the analytical precedures used in the work are given in a previous paper (Lockey, 1982). Hydrocarbons were isolated from the extracted lipids by column chromatography, using 200 x 20mm i.d. glass columns packed with alumina (Merck, neutral) and re-distilled petroleum spirit as the eluant. Before use, the alumina was heated at 110°C for 2 hr and transferred to petroleum spirit while still hot. Methylalkanes were separated from straight-chain components by refluxing each hydrocarbon mixture with Linde molecular sieve (type 5A) in iso-octane for 8 hr. Before use, the molecular sieve was heated at 380°C in a stream of nitrogen for 48 hr and then transferred to iso-octane while still hot (O'Connor et al., 1962). The methylalkane mixtures of both species were analysed with a model F17 Perkin-Elmer gas chromatograph using a 25 m vitreous silica capillary column with bonded phase BP1, 0.25/~m thick. The mixtures were temperature programmed from 161~300°C at 1.5°C/min. Helium at a flow rate of 3 ml/min was used as the carrier gas. Retention indices (I) were calculated from retention times which were determined by adding reference n-alkanes to each mixture and temperature programming from 160-300°C at 1.5°C/min (Kov~its, 1965).

RESULTS

Gas chromatograms of the cuticular hydrocarbons of B. orientalis (BO) and Periplaneta americana (PA) are given in Fig. 1. Methylalkanes account for approximately 78.9% and 39.8% of the cuticular hydrocarbons of B. orientalis and P. americana respectively and comprise terminally branched m o n o methylalkanes (BO, 18.0%; PA, 20.2%) and internally branched monomethylalkanes (BO, 52.6%; PA, 16.2%) and dimethylalkanes (BO, 8.2%; PA, 3.4%) (Table 1). Terminally branched monomethylalkanes are represented by 3-methylalkanes. 2-Methylalkanes were detected in neither mixture. Both species have homologous series of 3-methylalkanes ranging from 26 to 29 carbons (Table 1). A data system print-out of a MS scan of G C peak P A l lc, 3-methylhexacosane, which occurs at the base of the trailing edge of the large peak PA 11 b, cis,cis-6,9-heptacosadiene, is given in Fig. 2A. The MS scan shows a M-15 ion at m/z 365 (C26), an enhanced M-29 ion at m/z 351 (C/j) and ion doublets at m/z 56/7 (C4) and m/z 322/3 (C23).

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Cockroach methylalkanes

569

Table 1. Cuticularmonomethylalkanesand dimethylalkanesof Blatta orientalis and Periplaneta americana Blatta orientalis

Chain length Isomer C25 9, I1 & 13 5

C26

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3 5, 9 3, 7; 3, 9;&3, 11; 8,9,10,11,13&15 5 4 3 11 & 13 5 3

2579 2589 2610 2634 2655 2660 2673 2735 2758 2779

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5, 15;&5, 17;

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29

Internally branched monomethylalkanes occur in both species as continuous homologous series ranging from 26 to 30 carbons (Table 1). The series include 4-methyl- and 5-methylalkanes and a data system print-out of a MS scan of GC peak PAl la, 4-methylhexacosane, is given in Fig. 2B. The MS scan shows a M-15 ion at m/z 365 (C26), ion doublets at m/z 70/1 (C5) and m/z 308/9 (Cz2) and an enhanced fragment ion at m/z 337 (C24). The two species also have isomeric mixtures of monomethylalkanes and Fig. 2C gives a MS scan of GC peak PAl3, 9-, 11- & 13-methylheptacosane. The MS scan shows a molecular ion at m/z 394 (C28Hss) and ion doublets at each of the even-numbered ion clusters from 140/1 (CL0) to 280/1 (C20). Dimethylalkanes are present in both species in low proportions. All of the dimethylaikanes belong to type 2, which have their methyl side chains positioned at the beginning of the alkyl chain and separated by an odd and variable number of methylene groups (Lockey, 1985). Under GC analysis, type 2 dimethylaikanes elute close to a n-alkane with one carbon atom less and this is shown by GC peaks BO19 and BO21, which elute on either side of n-octacosane (BO20) (Fig. 1). Data system print-outs of MS scans of BO19 and BO21 are given in Fig. 3. The MS scan of BOl9 (Fig. 3A) shows a M-15 ion at m/z 393 (C28), ion doublets at m/z 84/5 (C6), m/z 140/1 (Cl0), m/z 168/9 (C12), m/z 196/7 (Cl4) and enhanced fragment ions at m/z 267 (Cl9) and m/z 351 (C25), which is interpreted as the fragmentation pattern of a mixture of 5, 15- and 5,17-dimethylheptacosane (Table 1). Although fragment ion m/z 239 (C~7) is of low abundance, the ion doublet at m/z 196/7 (C~4) is taken to indicate the presence of the 5,15-dimethylisomer, albeit in low abundance. The MS scan of BO21 (Fig. 3B) has a M-15 ion at m/z 393 (C28), an ion doublet at m/z 308/9 (C22) and enhanced fragment ions at m/z 127 (C9) and m/z 379 (C27), which

Isomer 9, 11 & 13 5 4 3

Retention GC peak Index(I) No. 2537 PA4 2554 5 2563 6 2575 7

3,7;3,9;3,11;&3,13; 2608 11, 12 & 13 2632

9 PAl0

4 3 9, 11 & 13 5 3 7, 11; 5, 11; 5, 13; 5, 15; & 5, 17; 3, 7; 12, 13& 14

2658 2675 2734 2753 2774 2761

lla lie PAl3 14 16 15

2784 2808 2840

17 19 PA22

4 3 13& 15

2853 2869 2936

23 24 PA27

5 4 3, 7;

2947 2957 3008

28 29 30

is interpreted as the fragmentation pattern of 3,7-dimethylheptacosane (Table 1). DISCUSSION

Previous workers identified the following cuticular hydrocarbons in B. orientalis (BO) and P. americana (PA): BO, nC27, 3-, 11- & 13-methylheptacosane (Jackson and Baker, 1970; Tartivita and Jackson, 1970): PA, nCl7 to nC29, 3-methylpentacosane, cis,cis-6,9-heptacosadiene and a small group of alkanes and alkenes in trace amounts ranging from 39 to 43 carbons (Baker et al. 1963; Beatty and Gilby, 1969; Gilby and Cox, 1963; Jackson, 1972). The present work shows that these analyses are incomplete. Both species have homologous series of 3-methylalkanes ranging from 26 to 29 carbons and isomeric mixtures of internally branched monomethylalkanes, ranging from 26 to 30 carbons, and dimethylalkanes with carbon numbers 27, 29 and 31 (Table 1). The present work also shows (Fig. 1) that B. orientalis has a continuous series of n-alkanes from nC23 to nC28 rather than nC27 alone (Tartivita and Jackson, 1970). The methylalkane mixtures of B. orientalis and P. americana show both qualitative and quantitative differences. The former include the presence in B. orientalis of the 5-, 6- & 7-methylisomers respectively of Cz6 (BO11), C2s (BO24) and C29 (BO28), which P. americana lacks, and the presence in the latter species alone of the 4-methylisomer of C25 (PA6) and the 4and 5-methylisomers of C29 (PA28 & PA29). Quantitative differences are more marked. In B. orientalis the monomethylisomers of C27 are the most abundant methylalkanes accounting for about 55.0% of the cuticular hydrocarbons. These isomers comprise the 3-methylisomer (BO18, 14.4%), the 5-methylisomer (BO16, 7.4%) and the 11- and 13-methylisomers (BOI5, 33.2%). In contrast, the most abundant and the second most abundant methylalkanes of P. a m e r -

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Cockroach methylalkanes

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11- & 13-methylheptacosane (PAl3, 7.0%) respectively (Table 1). The presence of homologous series of methylalkanes in the hydrocarbon mixtures of B. orientalis and P. americana brings their hydrocarbon compositions closer to those of other examined cockroach species (Lockey, 1980, 1985). This is particularly true C.B.P. 85/3B--E

of P. americana which, according to earlier workers, has a methylalkane mixture limited to 3-methylpentacosane and trace amounts of 3-methylhentetracontane (Jackson, 1972). Periplaneta japonica for example, has a methylalkane mixture comprising the 3-methylisomers of C27 and C29 and the 13-methylisomers of C27, C29 and C30 (Jackson, 1972), while the methylaikane mixtures of P. australasiae, P. brunnea

572

K . H . LOCKEYand B. DULARAY

and P. fuliginosa consist of the 3-methylisomers of C23 and C24 and a continuous series of the 11-, 12- and 13-methylisomers of C23 to C26 (Jackson, 1970). Arenivaga investigata, the desert cockroach, has a methylalkane mixture which consists of a complex isomeric mixture of internally branched m o n o methylalkanes, but which lacks 3-methylalkanes (Jackson, 1983). Apart from their higher mol. wt, the monomethylisomers of A. investigata are qualitatively very similar to those of B. orientalis and P. americana. However, A. investigata, in c o m m o n with other previously examined cockroach species, lacks dimethylalkanes. The dimethylalkanes of B. orientalis and P. americana are exclusively type 2 and in this respect they are unusual as types 1 and 2 usually occur together (Lockey, 1985). In other respects, the dimethylalkanes of the two species are unexceptional. As in B. orientalis and P. americana, the most commonly occurring type 2 dimethylalkanes are those which under G C analysis elute with nC26 , nC28 and nC~0, while the most c o m m o n dimethylisomers are those which have their first methyl side chain positioned at either carbon 3, 4 or 5 of the alkyl chain, (Lockey, 1985).

Acknowledgements--We are grateful to Professor A. A. Watson, Department of Forensic Medicine, University of Glasgow for use of V.G. Micromass gas chromatographmass spectrometer and to Dr. R. A. Anderson for the GC-MS analyses. We thank Mr. J. Laurie for helping to maintain insect stocks. The work was supported by SERC grant GR/CO 1351. REFERENCES

Baker G. L., Vroman H. E. and Padmore J. (1963) Hydrocarbons of the american cockroach. Biochem. biophys. Commun. 13, 360-365. Beatty I. M. and Gilby A. R. (1969) The major hydrocarbon of a cockroach cuticular wax. Naturwissenschaften 25, 373. Gilby A. R. and Cox M. E. (1963) The cuticular lipids of the cockroach, Periplaneta americana (L). J. Insect Physiol. 9, 671---681. Jackson L. L. (1970) Cuticular hydrocarbons of insects; 1I. Hydrocarbons of the cockroaches, Periplaneta austra-

lasiae, Periplaneta brunnea and Periplaneta fuliginosa. Lipids. 5, 38-41. Jackson L. L. (1972) Cuticular lipids of insects--IV. Hydrocarbons of the cockroaches, Periplaneta japonica and Periplaneta americana compared to other cockroach hydrocarbons. Comp. Biochem. Physiol. 41B, 331-336. Jackson L. L. (1983) Epicuticular lipid composition of the sand cockroach, Arenivaga investigata. Comp. Biochem. Physiol. 74B, 255-257. Jackson L. L. and Baker G. L. (1970) Cuticular lipids of insects. Lipids 5, 239-246. Kovfits E. sz. (1965) Gas chromatographic characterization of organic substances in the Retention Index System. Adv. Chromatogr. 1, 229-247. Lackie A. M. (1983) Immunological recognition of cuticular transplants in insects. Dev. Comp. Immunol. 7, 41-50. Lockey K. H. (1980) Insect cuticular hydrocarbons. Comp. Biochem. Physiol. 65B, 457-462. Lockey K. H. (1982) Hydrocarbons of adult Onymacris plana (P6ringuey) and Onymacris rugatipennis (Haag) (Coleoptera: Tenebrionidae). Insect Biochem. 12, 69-81. Lockey K. H. (1985) Insect cuticular lipids. Comp. Biochem. Physiol. 81B, 263-273. McCarthy E. D., Han J. and Calvin M. (1968) Hydrogen atom transfer in mass spectrometric fragmentation patterns of saturated aliphatic hydrocarbons. Analyt. Chem. 40, 1475-1480. Nelson D. R. (1978) Long-chain methyl-branched hydrocarbons: Occurrence, biosynthesis and function. Adv. Insect Physiol. 13, 1-33. Nelson D. R., Sukkestad D. R. and Zaylskie R. G. (1972) Mass spectra of methyl-branched hydrocarbons from eggs of the tobacco hornworm. J. Lipid Res. 13, 413-421. O'Connor J. G., Burrow F. H. and Norris M. S. (1962) Determination of normal paraffins in C20 and C32 paraffin waxes by molecular sieve adsorption. Analyt. Chem. 34, 82-85. Pomonis J. G., Fatland C. L., Nelson D. R. and Zaylskie R. G. (1978) Insect hydrocarbons: corroboration of structure by synthesis and mass spectrometry of mono- and dimethylalkanes. J. chem. Ecol. 4, 27-39. Pomonis J. G., Nelson D. R. and Fatland C. L. (1980) Insect hydrocarbons 2. Mass spectra of dimethylalkanes and the effect of number of methylene units between groups on fragmentation. J. chem. Ecol. 6, 965-972. Tartivita K. and Jackson L. L. (1970) Cuticular lipids of insects 1. Hydrocarbons of Leucophaea maderae and Blatta orientalis. Lipids 5, 35-37.