hydrocarbons of metriopus depressus (haag) and renatiella scrobipennis (haag) (coleoptera:...

Insect Biochem. Vol. 14, No. 1, pp. 65-75, 1984 0020-1790/84 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1984 Pergamon Press Ltd

HYDROCARBONS OF METRIOPUS DEPRESSUS (HAAG) AND RENATIELLA SCROBIPENNIS (HAAG)

(COLEOPTERA" TENEBRIONIDAE)

K. H. LOCKEY

Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.

(Received 28 March 1983)

Abstract--The hydrocarbons of Metriopus depressus (MD) and Renatiella scrobipennis (RS) have been analysed by gas chromatography and by combined gas chromatography-mass spectrometry and comprise n-alkanes (C23 to C3s, MD, 35%; RS, 78%), terminally branched monomethylalkanes (MD, C24 to C34, 36%: RS, C26 to C32, 3.0%) and internally branched monomethylalkanes (MD, C26 to C3~, 20%; RS, C26 to C32, 13%) and dimethylalkanes (MD, C29 to C37, 8%; RS, C27 to C35, 6~o).

The hydrocarbons of M. depressus and R. scrobipennis have in common with the hydrocarbons of five other species of tribe, Adesmiini, high proportions of C27 and C29 hydrocarbons, homologous series of 4-methyl and 5-methylalkanes and complex mixtures of C2s and C30 class D hydrocarbons which, on present evidence, characterizes tribe, Adesmiini.

Three species of genus, Onymacris can be distinguished from Physadesmia globosa and Stenocara graeilipes by having nC31 and higher proportions of the 3-methylisomers of C27 and C29 and from M. depressus and R. scrobipennis by having lower proportions of n-alkanes, 3-methylalkanes and 9-methylalkanes.

Key Word Index: Hydrocarbons, cuticle, chemotaxonomy, Tenebrionidae, Coleoptera

INTRODUCTION

The present work continues a detailed survey of the hydrocarbons of adult tenebrionid beetles to see to what extent taxa within family, Tenebrionidae can be characterized by hydrocarbon composition.

At the species level, each of the fourteen species now examined can be characterized by its hydrocarbons. Closely related species, such as congenerics, have qualitatively similar compositions, though the proportions in which the hydrocarbons occur can differ considerably. With more distantly related species, qualitative differences predominate (Lockey, 1981, 1982a,b,c).

Currently, species of tribe, Adesmiini (Gebien, 1937) are being examined to see if taxa above species level can be characterized in the same way. So far, the hydrocarbons of five species of Adesmiini, which in- habit the semi-arid to arid regions of western southern Africa (Koch, 1955; Penrith, 1975, 1979) have been analysed (Lockey, 1982a,b,c). Of the five species examined, the hydrocarbons of the three congeneric species, Onymacris plana, Onymacris rugatipennis and Onymacris marginipennis, share certain features such as the presence of nC31 and high proportions of the 3-methylisomers of C27 and C29, which distinguishes them from Physadesmia globosa and Stenocara gracilipes. At the same time, all five species have high proportions of the same C29 hydrocarbons, homologous series of 4-methyl and 5-methylalkanes and complex mixtures of the internally branched monomethylisomers of Czs and C3o (Lockey, 1982c).

Metriopus depressus and Renatiella scrobipennis are another two Adesmiine species from southern Africa. In the work to be described, their hydrocarbons, which

originate mainly from the cuticle, have been analysed by gas chromatography (GC) and by combined gas chromatography-mass spectrometry (GC-MS).

MATERIALS AND METHODS

The specimens for the work were collected in South West Africa/Namibia in May 1980. Adults of both species were collected during the day. M. depressus was found under stones on the gravel plains near Hentiesbaai, north of Swakopmund, while R. scrobipennis, which has a wide range of habitats, was collected at several locations in the central highlands, where it was found running among trees and shrubs (Koch, 1962; Penrith, 1975, 1979).

Details of the analytical procedure used in the work are given in a previous paper (Lockey, 1982a). The insects were kept alive until the time for lipid extraction when they were killed by freezing at -20°C and then refluxed with "AnalaR" chloroform for l hr. This procedure has been shown to extract 80~o of the hydrocarbons but less than 10~o of the more polar lipids which can be extracted from opened hard bodied beetles by prolonged (6 hr) refluxing with chloroform. Hydrocarbons were separated from the extracted lipids by column chromatography, using alumina and re-distilled petroleum spirit (boiling range 60 70°C) as the eluant.

The hydrocarbons were examined by i.r. spectroscopy as thin films between KBr plates to detect contaminants and to survey molecular composition.

The hydrocarbons were analysed with a model F17 Perkin-Elmer gas chromatograph, using a 24.5 m wall coated superior capacity open tubular (WSCOT) glass column coated with CP Sil 5. Both mixtures were analysed initially by temperature programming from 50 to 325°C at 5°C/min and subsequently from 160 to 325°C at T~C/min. Nitrogen at a flow rate of 3 ml/min was used as the carrier gas.

65

66 K. H. LOCKEY

Retention indices (/) were calculated from retention times, which were determined by adding even-numbered reference n-alkanes, ranging from C22 to C42, to each mixture and temperature programming from 160 to 325°C at 2°C/min (Ettre, 1964).

Mass spectra of the hydrocarbons of both mixtures were obtained with a 16F V.G. Micromass gas chromatograph- mass spectrometer fitted with the WSCOT column used in GC analyses. The mixtures were analysed by temperature programming from 200 to 300°C at 1 °C/rain. Helium at a flow rate of 1 ml/min was used as the carrier gas. The temperature of the ion source was 230°C and the ionization voltage was 28 eV. The mass spectrometer was interfaced to a V.G. 2000 data system and mass spectral scans (MS scans), ranging from m/z 20 to m/z 650, were taken repeti- tively at a cycle time of 8 sec. MS scans were selected for examination from the total ion count (TIC) chromatograms of the two mixtures. Background was subtracted automatic- ally by the data system before MS scans were examined. The mass spectra of branched alkanes were interpreted according to the criteria proposed by McCarthy et al. (1968), Nelson et al. (1972), Nelson (1978) and Pomonis et al. (1978, 1980).

Each mixture was examined for unsaturation by bromi- hating and then noting the disappearance of any component in subsequent GC analysis.

Straight-chain hydrocarbons were separated from branched hydrocarbons by refluxing each mixture with Linde molecular sieve in iso-octane (O'Connor et al., 1962).

RESULTS

Infra-red analyses showed both mixtures to comprise long-chain, aliphatic hydrocarbons, with some branched components. Like the five adesmiine species examined earlier (Lockey, 1982a,b,c), M. depressus and R. scrobipennis lack unsaturated hydrocarbons.

The gas chromatograms of the hydrocarbons of the two species are given in Figs 1A and 1B, while the approximate percentage composition, obtained by triangulation of GC peaks, and the identity of the components of each mixture, determined by GC MS, are given in Tables 1 and 2. Each percentage composition value is the average of three determinations, maximum variability - +0.93.

Both mixtures contain the following hydrocarbon classes: A, n-alkanes; C, terminally branched monomethylalkanes, D and E, internally branched monomethyl and dimethylalkanes respectively. The approximate percentage composition of each class is given in Fig. 5. Both mixtures form GC peaks containing components with different carbon numbers (Tables 1 and 2). These multi-component peaks consist of mixtures of dimethylisomers and either a 3-methylalkane, as in GC peak RS36, or a 5-methylalkane, as in GC peaks MD24, MD33 and RS35. The components of each of these peaks cannot be separated and they are estimated to occur in equal percentages (Tables 1 and 2).

Class A--n-alkanes

n-Alkanes account for about 35~ of the hydrocarbons of M. depressus and they form a continuous homologous series ranging from Cz3 to C33. A small percentage of nCas is also present in the mixture (MD44, 0.1~). nC29 is the most abundant member of the series (MD22, 16.9~), with nC2~ (MDll , 5.3~) and nC31 (MD31, 5.0% ), the next most abundant members.

R. scrobipennis has the highest percentage of n- alkanes (78~) of any of the tenebrionid species so far examined (Lockey, 1981, 1982a,b,c). The n-alkanes form a continuous homologous series, ranging from Cz3 to C3s, in which nCz7 is the most abundant member (RSll, 30.1~) and nC29 (RS22, 15.0~) and nC31 (RS32, 14.4~) are the next most abundant members of the series. In R. scrobipennis, as in the other examined adesmiine species, nCz8 (RS17) and nCao (RS28) form multi-component GC peaks with type 2 dimethylalkanes (Table 2).

Class C-- terminally branched monomethylalkanes

Class C~ hydrocarbons (2-methylalkanes) were not detected in either mixture. Both mixtures however, contain class C2 hydrocarbons (3-methylalkanes), which occur as incomplete homologous series.

dI is the difference between the retention index of a branched alkane and that of a n-alkane with the same carbon number and the dl values of the 3-methylalkanes of M. depressus range from 24 to 27, with an average value equal to 25. Those of R. scrobipennis range from 25 to 28, with an average value equal to 27.

The 3-methylalkanes of M. depressus account for about 36~o of the hydrocarbon mixture and range in carbon numbers from 24 to 34. 3-Methylnonacosane (MD25, 21.0°~) is the most abundant member of the series and indeed, of the whole hydrocarbon mixture (Table 1). MS scans of MD 25 (Fig. 2A) show the characteristic fragmentation pattern of a 3-methylalkane, with a M-29 ion of high abundance at m/z 393 (C2s), an enhanced M-57 ion cluster at m/z 364/5 (C26) and an enhanced C4 fragment ion at m/z 56. A M-15 ion shows at m/z 407 (C29).

GC peaks MD41 and MD42 are small peaks on the trailing edge of MD40 (Fig. 1 A) and their mass spectra were difficult to interpret. It proved impossible to identify MD42, while MD41 is identified tentatively as 3-methyltritriacontane (Table 1). Both the fragmentation pattern (Fig. 4C) and the d/value (25) of MD41 are consistent with the proposed identity. However, additional enhanced fragment ions occur in the MS scans of MD41, such as for example, the ion doublet at m/z294/5 (C21) and the fragment ion at m/z365 (C2o), which are unlikely to be derived from the preceding GC peak (MD40) and which cast doubt on the proposed identity.

The 3-methylalkanes of R. scrobipennis account for about 3~ of the hydrocarbon mixture and range in carbon number from 26 to 32. 3-Methylheptacosane (RSI5, 1.2~) is the most abundant member of the series.

In both species, 3-methylalkanes with even carbon numbers predominate (Fig. 5).

Class D--internally branched monomethylalkanes

Internally branched monomethylalkanes account for about 20 and 13~ of the hydrocarbon mixtures of M. depressus and R. scrobipennis respectively. M. depressus has a complete homologous series of class D hydrocarbons ranging in carbon number from 26 to 34 as well as a small percentage of the monomethylisomers of C35 (MD45, 0.2~). The most abundant members of the series are the monomethylisomers of C27 (MD12 and MD13, 5.6~) and C29 (MD23 and MD24, 5.7~,,,). Similarly, the class D hydrocarbons of

Hydrocarbons of M. depressus and R. scrobipennis 67

(A ) M. depressus 25

nC3~ nC3~

47 40

I I I 70 60 50

([~) R. $cl*oblpertn ls nC

nC~ i ~. 3 2 ~ 2 2

nC33

\ n C~s 3 9

t t I 70 60 50

(C) R. scrob/penms ( branched alkanes)

22 nC29

nC27

nCe~ l

I l I I 40 30 20 I0

11 nC27

I ! I I 40 30 20 I0

Retention time (min)

42 4 1 ~ L ~ l M I I L ~ , 3 2 i I I I I I | I

nC3~ nC~ nO3, .C=, nC~r nC~ / nC=3 "~s4 n032 ~ s o ~ nClti nCz4

Fig. 1. Gas chromatograms of the hydrocarbons of Metriopus depressus (A), Renatiella scrobipennis (B) and the branched alkanes of R. scrobipennis (C), the n-alkanes having been removed by molecular sieving. GC analyses on a 24.5 m WSCOT glass column, coated with CP Sil 5 and temperature

programmed from 160 to 325°C at 2°C/min. Carrier gas: nitrogen at 3 ml/min.

68 K. H. LOCKEY

Table 1. Hydrocarbons of Metriopus depressus

GC Retention Carbon ~o peak no. index (/) No. d l Class Composition Hydrocarbon

MD 1 2 3 4 5 6 7

2300 2373 2400 2500 2540 2574 2600

8 2638 9 2660

10 2673 11 2700 12 2740 13 2752

14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32 33

2775 2783 2800 2809 2836 2849 2860 2876 2900 2936 2953

2975 3000 3010 3029 3060 3075 3100 3130 3157

34 3176 35 3200 36 3213 37 3260 38 3300 39 3333 40 3363 41 3375 42 3385 43 3458 44 3500 45 3529 46 3558 47 3584

23 A 0.1 24 27 C2 0.1 24 -- A 0.1 25 -- A 1.3 26 60 D 0.1 26 26 C2 0.6 26 -- A 0.2 27 62 D 0.2 27 40 D 0.1 27 27 C2 0.2 27 -- A 5.3 28 60 D 4.4 28 48 D 0.6* 29 148 E 0.6* 28 25 C2 6.5 29 117 E 0.4 28 -- A 2.5 29 91 E 0.7 29 64 D 0.8 29 51 D 0.2 29 40 D 2.8 29 24 C2 0.8 29 -- A 16.9 30 64 D 4.7 30 47 D 1.0" 31 147 E 1.0" 30 25 Cz 21.0 30 A 2.5 31 90 E 1.3 31 71 D 0.8 31 40 D 2.2 31 25 Cz 0.7 31 A 5.0 32 70 D 0.8 32 43 D 0.5* 33 143 E 0.5* 32 24 C2 6.0 32 -- A 0.5 33 87 E 0.6 33 40 D 0.6 33 -- A 0.2 34 67 D 0.3 35 137 E 1.7 34 25 C2 0.3

0.4 36 142 E 0.2 35 -- A 0.1 36 71 D 0.2 37 142' E 1.0

0.4

n-Tricosane 3- Methyltricosane n-Tetracosane n-Pentacosane 9- Methylpentacosane 3- Methylpentacosane n-Hexacosane 9- Methylhexacosane 4-Methylhexacosane 3- Methylhexacosane n- Heptacosane 7-, 9-, I 1- and 13-Methylheptacosane 5- Methylheptacosane 9,13-Dimethylheptacosane 3-Methylheptacosane 5,9- and 5,1 l-Dimethylheptacosane n-Octacosane 3,7-, 3,9- and 3,11-Dimethylheptacosane 9-, 10-, 12-, 13- and 14-Methyloctacosane 6-Methyloctacosane 4-Methyloctacosane 3-Methyloctacosane n-Nonacosane 7-, 9-, 11-, 13-and 15-Methylnonacosane 5-Methylnonacosane 11,15-Dimethylnonacosane 3-Methylnonacosane n-Triacontane 3,7-, 3,9- and 3,11-Dimethylnonacosane 12-, 14- and 15-Methyltriacontane 4-Methyltriacontane 3-Methyltriacontane n-nentriacontane 7-, 9-, 11-, 13-and 15-Methylhentriacontane 5-Methylhentriacontane 11,15- and 13,17-Dimeth~,lhentriacontane 3- Methylhentriacontane n-Dotriacontane 3,7-, 3,9- and 3,1 l-Dimethylhentriacontane 4-Methyldotriacontane n-Tritriacontane 7-, 9-, 11-, 13-and lfi-Methyltritriacontane I 1,21-, 13,17- and 15,19-Dimethyltritriacontane 3-Methyltritriacontane

11,15- and 13,17-Dimethyltetratriacontane n-Pentatriacontane 11-, 13-and 15-Methylpentatriacontane 13,17- and 15,19-Dimethylpefitatriacontane

*Estimate.

R. scrobipennis form a complete, though less extensive, series which ranges in carbon n u m b e r f rom 26 to 32. As in M. depressus, the mos t a b u n d a n t members of the series are the monomethyls iomers of C27 (RS12 a n d RS13, 5 .1~) and C29 (RS23 and RS24, 5.2~).

Like the adesmiine species examined earlier (Lockey, 1982a,b,c), M. depressus and R. scrobipennis have complex mixtures of class D hydrocarbons , consisting of homologous series of 4-methyl and 5-methylalkanes as well as homologous series of isomeric mixtures,

such as for example, 7-, 9-, 11- and 13-methylheptacosane ( M D I 2 and RS12).

In bo th species, the 4-methylalkanes have odd carbon numbers and the 5-methylalkanes have even carbon numbers . One exception, 5-methylhexacosane (RS10), occurs in R. scrobipennis. MS scans of the small G C peak RS10 (Fig. 1B), which is identified as a mixture of the 5-methyl and the 6-methyl isomer of C26, are given in Fig. 3. Early MS scans of the peak (Fig. 3A) show the f r agmenta t ion pa t te rn o f the 6-

Hydrocarbons of M. depressus and R. scrobipennis

Table 2. Hydrocarbons of Renatiella scrobipennis

69

GC Retention Carbon ~o peak no. index (/) No. d l Class Composition Hydrocarbon

RS 1 2300 23 -- A <0.1 n-Tricosane 2 2400 24 -- A < 0.1 n-Tetracosane 3 2500 25 -- A 4.8 n-Pentacosane 4 2538 26 62 D 0.6 7-, 9- and 11-Methylpentacosane 5 2550 26 50 D 0.4 5-Methylpentacosane 6 2572 26 28 C2 0.4 3-Methylpentacosane 7 2588 27 112 E 0.4 5,9-, 5,11-, 5,13-, 5,15- and 5,17-Dimethylpentacosane 8 2600 26 -- A 2.4 n-Hexacosane 9 2635 27 65 D 0.2 8-, 9-, 10-, 11-, 12- and 13-Methylhexacosane

10 2648 27 52 D 0.1 5- and 6-Methylhexacosane 11 2700 27 -- A 30.1 n-Heptacosane 12 2738 28 62 D 3.4 7-, 9-, 11- and 13-Methylheptacosane 13 2754 28 46 D 1.7 5-Methylheptacosane 14 2765 29 135 E 0.7 9,13- and 11,15-Dimethylheptacosane 15 2773 28 27 C2 1.2 3-Methylheptacosane 16 2783 29 117 E 1.2 5,13-, 5,15- and 5,17-i)imethylheptacosane 17 2800 28 -- A 2.5 n-Octacosane

29 100 E 0.5 3,9- and 3,15-Dimethylheptacosane 18 2832 29 68 D 0.5 10-, 11-, 12-, 13- and 14-Methyloctacosane 19 2848 29 52 D 0.1 6- and 8-Methyloctacosane 20 2861 29 39 D 0.3 4-Methyloctacosane 21 2875 29 25 C2 0.I 3-Methyloctacosane 22 2900 29 -- A 15.0 n-Nonacosane 23 2934 30 66 D 4.9 7-, 9-, 11-, 13- and 15-Methylnonacosane 24 2952 30 48 D 0.3 5-Methylnonacosane 25 2961 31 139 E 0.6 7,11-, 9,13-, 11,15- and 13,17-Dimethylnonacosane 26 2973 30 27 C2 0.8 3-Methylnonacosane 27 2982 31 118 E 1.0 5,9-, 5,11-, 5,13-, 5,15- and 5,19-Dimethylnonacosane 28 3000 30 -- A 0.9 n-Triacontane

31 100 E 0.3 3,11-, 3,13-, 3,15- and 3,17-Dimethylnonacosane 29 3031 31 69 D 0.1 10-, 11-, i2-, 13-, 14- and 15-Methyltriacontane 30 3042 31 58 D 0.2 8-Methyltriacontane 31 3064 31 36 D 0.3 4-Methyltriacontane 32 3100 31 -- A 14.4 n-Hentriacontane 33 3132 32 68 D 0.3 11-, 13- and 15-Methylhentriacontane 34 3144 32 56 D 0.1 7-Methylhentriacontane 35 3153 32 47 D <0.1" 5-Methylhentriacontane

33 147 E <0.1" 9,13-, 11,15- and 13,17-Dimethylhentriacontane 36 3174 32 26 C2 0.4* 3-Methylhentriacontane

3181 33 119 E 0.4* 5,13-, 5,15- and 5,17-Dimethylhentriacontane 37 3200 32 -- A 1.2 n-Dotriacontane 38 3264 34 136 E 0.1 11,15-, 13,17- and 14,18-Dimethyldotriacontane 39 3300 33 -- A 5.6 n-Tritriacontane 40 3379 35 121 E 0.3 5,17- and 5,19-Dimethyltritriacontane 41 3400 34 -- A 0.4 n-Tetratriacontane 42 3500 35 -- A 0.7 n-Pentatriacontane

*Estimate.

methylisomer, consisting of a molecular ion at m/z 380 (C27Hs6), an ion double t a t m/z 280/1 (C20) and enhanced f ragment ions at m/z98 (C7) and m/z309 (C22). Later MS scans of the peak (Fig. 3B) show the same molecular ion, bu t a different f ragmenta t ion pat tern, which now consists of an ion doublet at m/z 294/5 (C21) and enhanced f ragment ions at m/z84 (C6) and m/z 323 (C23) and which is interpreted as being that of the 5-methylisomer of C26.

Homologous series of 7-methylalkanes occur in all of the adesmiine species so far examined, bu t only O. rnarginipennis (Lockey, 1982c) and R. scrobipennis have 7-methylalkanes in amounts sufficient to form single-component peaks, such as for example, the

small G C peak RS34 (Table 2). MS scans of RS34 (Fig. 2B) show a M-15 ion at m/z435 (C31), ion doublets at m/z 112/3 (Cs) and m/z 336/7 (C24) and an enhanced ion cluster at m/z 364/5 (C26), which is interpreted as the f ragmenta t ion pat tern of 7-methyl- hent r iacontane (Table 2).

A characteristic of M. depressus is the presence of s ingle-component G C peaks of 9-methylalkanes (MD5 and MD8).

Class E--internally branched dimethylalkanes

Both species have low percentages of dimethylalkanes (MD, 8 ~ ; RS, 6~) . The incomplete homologous series of M. depressus ranges in ca rbon number

70 K.H. LOCKEY

( A ) ~oo -

9 0 -

8 0

7 0

6 0

50

4 0

30

2 0

8 7

9 0 -

8 0 -

7 0 -

6 0 -

5 0 -

4 0

3 0

2 0

I 0

5 7

,6

I 0 0 57

I

I I 4 0 6 0 8 0 I 0 0 20

M D 2 5 3- met hylnonacosane Scan N 0 4 7 5 56

c l I

C 2 - - C - - CZ6 I I 393 3 6 4 / 5

3 9 3

,J ,,, i J l J , , , , 2 0 0

, I "I ..I .I J i

30O

36'11 4 0 7 M - 1 5

t i i i 4 0 0

RS 34 7 - methy l hen t r i a con tone

Scan N 0 , 3 8 9 112

c l I

c~-- C --C2a I I 365 3 3 6 / 7

i i i i i l I / I I / r 1 I i |

4 0 6 0 8 0 2 0 0 20 4 0 6 0 8 0 3 0 0 2 0 4 0 6 0 8 0 4 0 0 20 4 0

m / z

Fig. 2. MS scans of GC peaks MD25 (A) the main hydrocarbon of M. depressus and RS34 (B). Scans Nos 475 and 389. The latter scan is complicated by presence of ion doublets from the preceding mixture

of monomethylisomers.

from 29 to 37, with the dimethylisomers of C29 the most abundant members of the series (MD24 and MD27, total of 2.3%). R. scrobipennis also has an incomplete series which ranges in carbon number from 27 to 35, with the dimethylisomers of C2~ in the highest abundance (RS14, RSI6 and RSI7, total of 2.4%).

Like the other adesmiine species examined earlier, M. depressus and R. scrobipennis have type 1 and type 2 dimethylalkanes. The side chains of the former occur near the centre of the chain and are always separated by three methylene groups, while those of the latter occur near the end of the chain and are separated by an odd number of methylene groups (Lockey, 1982a).

Type 1 dimethylalkanes have been found in many insect species (Blomquist and Jackson, 1979; Jackson and Blomquist, 1976; Lockey, 1980; Nelson, 1978) and they are represented in M. depressus by GC peaks MD13, MD24, MD33, MD40, MD43 and MD46 and in R. scrobipennis by GC peaks RS14, RS25, RS35 and RS38 (Tables 1 and 2).

GC peak MD40 of the above series however, is unusual in that it contains an 11,21-dimethylisomer of C33 , which although showing internal branching, has its methyl side chains separated by nine rather than

the usual three methylene groups of type I dimethylalkanes. MS scans of the leading edge of MD40 (Fig. 4A) show a M-15 ion at m/z 477 (C34), ion doublets at m/z 196/7 (C14), m/z 224/5 (C16) and m/z 252/3 (C]8) and enhanced fragment ions at m/z 267 (C19), m/z 295 (C20 and m/z 323 (C23), which is interpreted as the fragmentation pattern of a mixture of the 13,17- and 15,19-dimethylisomers of C33. These are typical type 1 dimethylalkanes. However, later MS scans of the apex of MD40 (Fig. 4B) show the same M-15 ion but a different fragmentation pattern, consisting of ion doublets at m/z 168/9 (C12) and m/z 196/7 (C]4) and enhanced fragment ions at m/z295 (C21), m/z 323 (C23) and m/z 351 (C2 s). The absence of ion doublets at m/z 252/3 (C18) and 280/1 (C2o) indicates that this fragmentation pattern is not that of a mixture of the more usual 11,15- and 13,17-dimethylisomers of C33, which are likely to fragment in the following way:

168 239 196 267

c I c I c I c I I I I I

C l o - C -- C 3 - C C18 C12 C - C 3 C C16

I I i I 351 280 323 252


(A) boo 57 98

9 0 RSIO 6 - methylhexacosane C ] Scan No. 192 I

80 - - C C5 I ~- c2° n

7 0 3 0 9 2 8 0 / I

6 0

50 X5 m

40

308

30

c

u u I 0 0 - O0 2 0 0 300 4 0 0

JO ,~ 57

9 0 - RSIO 5 - methy lhexacosone 8 4

Scan No. 194 C (B) 8o - i C 4 - C - - C2/

7 O - I t

.323 2 9 4 / 5 6 0 -

X5 L 5 0 -

4 0 3 2 3

ILl 111 i I , ,, 294 :580 M ÷

I I I I I I I I I I F I

4 0 6 0 80 O0 2 0 4 0 6 0 8 0 2 0 0 20 40 60 flO 3 0 0 20 4 0 6 0 80 4 0 0

m / z

Fig. 3. MS scans of GC peak RS10. Scan Nos 192 (A) and 194 (B). Scan complicated by presence of ion doublets from the preceding mixture of monomethylisomers.

Similarly, the absence of ion doublets at rn/z 224/5 (C~6) and rn/z 252/3 (C1 a) indicates that the fragmentation pattern is probably neither that of a mixture of the 11,17- and 13,19-dimethylisomers nor that of a mixture of the 11,19- and 13,21-dimethylisomers of C33 . Accordingly, the fragmentation pattern is interpreted as being that of the 11,21-dimethylisomer of C33 , though this interpretation assumes that the enhanced fragment ion at m/z 295 (C2~) derives from the 15,19-dimethylisomer, which elutes earlier (Fig. 4A).

Type 2 dimethylalkanes elute with or close to a n- alkane having one carbon atom less (Nelson et al., 1980) and they can be revealed by removing the n- alkanes of a hydrocarbon mixture by molecular sieving. The type 2 dimethylalkanes of M. depressus elute close to nC2s (MD15 and MDI7), nC30 (MD27) and nC32 (MD36). Those of R. scrobipennis (Fig. 1C) form a more extensive series, the members of which elute close to or with all of the even-numbered n-alkanes f r o m n C 2 6 t o n C 3 4 . Type 2 dimethylalkanes account for about 3 and 4% respectively of the class E hydrocarbons of M. depressus and R. scrobipennis.

DISCUSSION

Although the hydrocarbon mixture of R. scrobipennis consists mainly of n-alkanes (Fig. 5), it is qualitatively very similar to the mixtures of M. depressus and the other adesmiine species already examined (Fig. 6). For example, 61 and 65~o of the hydrocarbons of M. depressus and O. plana (Lockey, 1982a) occur in R. scrobipennis, while 52 and 54~ o of the hydrocarbons of the latter species are found in M. depressus and O. plana respectively. Like M. depressus and the other adesmiine species, R. scrobipennis has homologous series of 4-methyl and 5-methylalkanes and complex mixtures of C28 and C3o class D hydrocarbons, though unlike the other species, R. scrobipennis has Cz7 hydrocarbons in higher proportions than C29 hydrocarbons.

The n-alkane/branched alkane ratio of R. scrobipennis equals 3.5, which is the highest ratio encountered in the present survey and which is in contrast to an average value equal to 1.0 for all tenebrionid species examined in the survey (Lockey, 1981, 1982a,b,c). The

IB 14/I -E

72

(A) loo 57

90

80

70

6O

50

40

30

.20

tO

(BI Joo~- 57

9 0 -

8 0 -

" 7 0 -

6 0 - -

5 0

IO

i i

(C)1oo:- 57

9 0 -

8 0 -

7 0 -

6 0 -

5O

4 0 56

3 0

2 0

IO

K. H. LOCKEY

IO0

M D 4 0 13,17-and 15,19-dimethylfrifriocontane S~on NO, 753

X5

2 6 7

c r r

MO40 11,.21- dimethyltritriocontone Scan N o . 7 5 7

; f i

2 0 0

196 2 6 7

c l c l I I

Ci2-- C - - C¢ T C Cle 1 3 2 3 2 5 2

2 9 5

3 2 3

,JIIt ;'00 r I r r

X5

I 0 0 .200

I iI i i

3 0 0

323

351

d I i

2 2 4 .29,5

e l c l I I

Ci4-- C - - C ~ C - - C j 4 I ]

2 9 5 2.24

4 7 7 M-15

llhl IL 4bo ' ' ' 5~o

168 5'23

c l c l I i

Clo-- C - - C9-.-~.- C - - C t z /

351 196

ill JlJ 4 0 0

MD4/ 3-methyltrJtriacontane Scan No.760

X 5

4 0 6 0 80 4 0 ,~o 2'o 4o ;,o ;o .2~,o 2'° II11,

,'o ,,'o 3~o .2°

56

cl I

c~ -cTC ,o 4 4 9 4 2 0 / I

4 4 9

Jl j 1,,oi i , i i /

4 0 6 0 8 0 4 0 0 20 4 0 6 o

m / z

Fig. 4. MS scans of GC peaks MD40 (A and B) and MD41 (C). Scan Nos 753,757 and 760.

4 7 7 M - / 5

lJ

4 7 8 M +

!, 0 5OO

reason for this high ratio is not known (Hadley and Louw, 1980; Jackson et al., 1980). High n-alkane/ branched alkane ratios occur in a few other coleo- pteran species such as Lepidochora discoidalis (15.0), a tenebrionid beetle which inhabits the sand-dunes of the Namib desert (Hadley and Louw, 1980), adult and larva A ttagenus megatoma (15.2 and 14.9 respectively), the black carpet beetle (Baker et al., 1979a) and larva Lasioderrna serricorne (5.6), the cigarette beetle. The

adult of the latter however, has a ratio as low as 0.2 (Baker et al., 1979b).

In the case of R. scrobipennis, Penrith (1979) notes that this species shows more primitive characters than other species of the tribe, Adesmiini and it may be that a high n-alkane/branched alkane ratio is a primitive feature of the tribe. A similar relationship is shown by congeneric species of Tenebrio and Tribolium (Lockey, 1978a,b), which have the highest n-alkane/branched


Chain length 20 22 24 26 28 30 32 34 36 38 4c

20 22 24 26 28 30 32 34 36 :38 40

Choin length

Fig. 5. Percentage composition of the hydrocarbons of Metriopus depressus (MD) and Renatiella scrobipennis (RS). Class A, n-alkanes; C2, terminally branched monomethylalkanes; D and E, internally

branched monomethyl and dimethylalkanes respectively.

ratios after that of R. scrobipennis, namely, 2.6 adult Tenebrio molitor; 24.0, larva T. molitor (Bursell and Clements, 1967) and 1.6, adult Tenebrio obscurus, Tribolium castaneum and Tribolium confusum, and which, like R. scrobipennis, may be considered to be among the least advanced of their respective tribes.

In earlier work (Lockey, 1982c), the presence of nC3~ and high proportions of the 3-methylisomers of C27 and C29 in the hydrocarbon mixtures of the congeneric species, O. plana, O. rugatipennis and O. marginipennis was used to distinguish genus, Onymacris from Physadesmia globosa and Stenocara gracilipes. However, both M. depressus and R. scrobipennis have

nC31 and appreciable percentages of C31 hydrocarbons (MD 13.4%; RS, 15.6%) so that clearly, these characters are not exclusive to the congeneric species of Onymacris.

Because of its high proportion of n-alkanes, there is little difficulty in distinguishing R. scrobipennis from the congeneric species of Onymacris. However, sepa- rating M. depressus from these three species is more difficult, for not only does M. depressus have nC3a and appreciable proportions of C3~ hydrocarbons, but it also has relatively high percentages of the 3- methylisomers of C27 (MD14, 6.5%) and C29 (MD25, 21.0%) (Fig. 6). As a result, M. depressus has to be

74 K.H. LOCKEr

train Imgth 23 2 5 2 7 2 9 31 3 3 % H y d r o c o r b o n A D 5 4 3 E I A D 5 4 3 E A D 5 4 3 E A D 5 4 3 E A O 5 4 3 E A D 5 4 3 E Con~

! S p e c i e s

3 0 -

OP 2 o -

I 0 -

3 0 -

OR 2 0 -

10 -

3 0 -

O M

2 0 -

f O -

3 0 -

P G L

2 0 -

I 0 -

3 0 -

SG 2 0 -

I 0 -

3 0 -

MD 2 0 -

I 0 -

3 0 -

RS 2 0 -

I 0 -

0 7 2 6 [ 5 I 4214 2513 [ 5 8 8

0.3 4 . 4 64.7 16 .9 1.6 8 8

O . I f . 4 35.6 4 0 . 3 4 .2 8 2

0 2 0 9

O I 2 .9

7.8 . 5.9 9 0

2 3 3 353 f .4 0 .3 6 3

0 .2 2 .0 18.5 4 5 , 9 13.4 2.5 8 3

6 . 6 3 8 . 3 2 2 . 9

7 i /

1 5 . 6 5.9 8 9

Fig. 6. Percentage composition of the major hydrocarbons of seven species of tribe, Adesmiini. OP, Onymacris plana; OR, Onymacris rugatipennis; OM, Onymacris marginipennis; PGL, Physadesmia globosa ; SG, Stenocara gracilipes; MD, Metriopus depressus ; RS, Renatiella scrobipennis. A, n-alkane; D, monomethylisomer mixtures; 5, 4, and 3, respectively, 5-methyl-, 4-methyl- and 3-methylisomers;

E, dimethylisomer mixtures.

distinguished from the congeneric species of Ony- macris by other characteristics of its hydrocarbon mixture. These include higher percentages of nC27, nC2o and nC31, 3-methylalkanes and the 9-methylisomers of C2 s and C2~.

In spite of these differences, the hydrocarbons of the seven species of tribe, Adesmiini (Fig. 6) share certain

characteristics, such as high proportions of C27 and C29 hydrocarbons, homologous series of 4-methyl and 5-methylalkanes and complex mixtures ofC28 and C3o class D hydrocarbons, which, on present evidence, can be used to characterize the tribe. It is more difficult to characterize genus, Onymacris and different sets of c- f latters are required to distinguish the genus from


P. globosa and S. gracilipes on the one hand and from M. depressus and R. scrobipennis on the other.

Acknowledgements--I am most grateful to Dr M-L Penrith and Mr S. Louw, State Museum, South West Africa/ Namibia, who chose the collecting sites and who identified and helped to collect specimens. I thank the University Court, University of Glasgow, for granting me study leave and the Carnegie Trust for the Universities of Scotland for financial help. I am indebted to Professor H. E. Paterson, Department of Zoology, University of the Witwatersrand, South Africa, who provided laboratory accommodation and to Mr C. C. Coetzee, Director, State Museum, SWA/ Namibia, who provided study facilities. I thank Professor W. A. Harland, Department of Forensic Medicine, Univer- sity of Glasgow, for use Of the V. G. Micromass gas chromatograph-mass spectrometer and Dr R. A. Anderson for GC-MS analyses. Mrs F. Lawrie kindly supplied and interpreted the infra-red spectra.

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Baker J. E., Nelson D, R. and Fatland C. L. (1979a) Developmental changes in cuticular lipids of the black carpet beetle, Attagenus megatoma. Insect Biochem. 9, 335-339.

Baker J. E., Sukkestad D. R., Nelson D. R. and Fatland C. L. (1979b)Cuticular lipids of larvae and adults of the cigarette beetle, Lasioderma serricorne. Insect Biochem. 9, 603~611.

Blomquist G. J. and Jackson L. L. (1979) Chemistry and biochemistry of insect waxes. Prog. Lipid Res. 17, 319- 345.

Bursell E. and Clements A. N. (1967) The cuticular lipids of the larva of Tenebrio molitor L (Coleoptera). J. Insect Physiol. 13, 1671-1678.

Ettre L. S. (1964) The kovhts retention index system. Analyt. Chem. 36, 31A-40A.

Gebien H. (1937) Katalog der Tenebrioniden. Teil 1. Pubbl. Mus. ent. Pietro Rossi Duino. 2, 505-883.

Hadley N. F. and Louw G. N. (1980) Cuticular hydrocarbons and evaporative water loss in two tenebrionid beetles from the Namib desert. S. Aft. J. Sci. 76, 298-301.

Jackson L. L. and Blomquist G. J. (1976) Insect Waxes. In Chemistry and Biochemistry of Natural Waxes (Ed. by Kolattukudy P. E.), 1st edn, pp. 201-233. Elsevier, Amsterdam.

Jackson L. L., Hadley N. F. and Blomquist G. J. (1980) Epicuticular lipids of the desert tenebrionid beetle, Eleodes armata: identification of the branched hydrocarbons. Insect Biochem. 10, 399-402.

Koch C. (1955) Monograph of the Tenebrionidae of Southern Africa. 1. Tentyriinae, Molurini, Trachynotina, Somaticus Hope. Transv. Mus. Mem. 7, 1-242.

Koch C. (1962) The Tenebrionidae of Southern Africa.

XXXI. Comprehensive notes on the tenebrionid fauna of the Namib desert. Ann. Transv. Mus. 24, 61-106.

Lockey K. H. (1978a) The adult cuticular hydrocarbons of Tenebrio molitor L and Tenebrio obscurus F. (Coleoptera: Tenebrionidae). Insect Biochem. 8, 237-250.

Lockey K. H. (1978b) Hydrocarbons of adult Tribolium castaneum Hbst. and Tribolium confusum Duv. (Coleo- ptera: Tenebrionidae). Comp. Biochem. Physiol. 61B, 401-407.

Lockey K. H. (1980) Insect cuticular hydrocarbons. Comp. Biochem. Physiol. 65B, 457-462.

Lockey K. H. (1981) Cuticular hydrocarbons of adult Cylindrinotus laevioctostriatus (Goeze) and Phylan gibbus (Fabricius) (Coleoptera: Tenebrionidae). Insect Biochem. 11,549-561.

Lockey K. H. (1982a) Hydrocarbons of adult Onymacris plana (P~ringuey) and Onymacris rugatipennis (Haag) (Coleoptera: Tenebrionidae). Insect Biochem. 12, 69-81.

Lockey K. H. (1982b) Hydrocarbons of adult Physadesmia globosa (Haag) and Stenocara gracilipes (Haag) (Coleo- ptera: Tenebrionidae). Insect Biochem. 12, 331-342.

Lockey K. H. (1982c) Cuticular hydrocarbons of adult Onymacris marginipennis (Brrme) (Coleoptera: Tene- brionidae). Comp. Biochem. Physiol. 73B, 275-282.

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.

Nelson D. R., Fatland C. L., Howard R. W., McDaniel C. A. and Blomquist G. J. (1980) Re-analysis of the cuticular methylalkanes of Solenopsis invicta and S. richteri. Insect Biochem. 10, 409~118.

O'Connor J. G., Burrow F. H. and Norris M. S. (1962) Determination of normal paraffins in C20 to C32 paraffin waxes by molecular sieve adsorption. Analyt. Chem. 34, 82-85.

Penrith M-L. (1975) The species of Onymacris Allard (Coleoptera: Tenebrionidae). Cimbebasia 4, 48-97.

Penrith M-L. (1979) Revision of the western southern african Adesmiini (Coleoptera: Tenebrionidae). Cimbe- basia 5, 3-94.

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 the number of methylene units between methyl groups on fragmentation. J. chem. Ecol. 6, 965-972.

hydrocarbons of metriopus depressus (haag) and renatiella scrobipennis (haag) (coleoptera:...

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