Semiochemicals of the Scarabaeinae: VI. Identification of EAD-active constituents of abdominal secretion of male dung beetle, Kheper nigroaeneus.

Article abstract of DOI:10.1023/A:1014583826875

Using gas chromatography with flame ionization detection (FID) and electroantennographic detection (EAD) in parallel, and employing chiral and achiral capillary columns, three constituents of the abdominal sex-attracting secretion of male Kheper nigroaeneus dung beetles were found to elicit reproducible EAD responses in male and female K. nigroaeneus antennae. One of these constituents is present in the secretion in such a small quantity that it could not be detected by FID, and it was not identified. The other constituents were identified as 3-methylindole (skatole) and (R)-(+)-3-methylheptanoic acid.

Full text of DOI:10.1023/A:1014583826875

Journal of Chemical Ecology, Vol. 28, No. 3, March 2002 (�C 2002)
SEMIOCHEMICALS OF THE SCARABAEINAE: VI.
IDENTIFICATION OF EAD-ACTIVE CONSTITUENTS
OF ABDOMINAL SECRETION OF MALE DUNG
BEETLE, Kheper nigroaeneus
�
B. V. BURGER and W. G. B. PETERSEN
Laboratory for Ecological Chemistry
Department of Chemistry
University of Stellenbosch
Stellenbosch 7600, South Africa
(Received July 10, 2001; accepted November 19, 2001)
Abstract—Using gas chromatography with flame ionization detection (FID)
and electroantennographic detection (EAD) in parallel, and employing chiral
and achiral capillary columns, three constituents of the abdominal sex-attracting
secretion of male Kheper nigroaeneus dung beetles were found to elicit repro-
ducible EAD responses in male and female K. nigroaeneus antennae. One of
these constituents is present in the secretion in such a small quantity that it could
not be detected by FID, and it was not identified. The other constituents were
identified as 3-methylindole (skatole) and (R)-(+)-3-methylheptanoic acid.
Key Words—Scarabaeinae, Kheper nigroaeneus, dung beetle, sex attractant,
sex pheromone, insect semiochemicals, EAD, electroantennographic detection.
INTRODUCTION
The coprophagous fauna associated with the mammals of Africa plays an important
role in the recycling of nutrients and in the destruction of the habitat of many dung
breeding flies (Heinrich and Bartholomew, 1979). During the summer months
when dung beetle activity reaches its peak, hardly any flies are found in game
reserves with normal herbivore populations. In contrast, areas a few kilometers
outside these reserves, where the cultivation of land or other farming practices
have resulted in a decrease in the dung beetle population, the indigenous people
areoftenplaguedbyswarmsofflies. SimilarproblemsareexperiencedinAustralia,
�
To whom correspondence should be addressed. E-mail: lecus@maties.sun.ac.za
5
01
0
098-0331/02/0300-0501/0 �C 2002 Plenum Publishing Corporation

5
02
BURGER AND PETERSEN
where cattle were introduced without the associated insect fauna, resulting in the
deterioration of pastures (Waterhouse, 1974) and the uncontrolled increase of the
fly population (Hughes, 1970).
Based on their nesting behavior, dung beetles of the subfamily Scarabaeinae
can be divided into three groups (Halffter and Matthews, 1966; Bornemissza,
1969). The paracoprids construct their nests under the dung pad by excavating
tunnels in which dung is packed, the endocoprids excavate a chamber in the dung
pad itself, forming brood-balls within this chamber, and the telecoprids detach a
portion of dung from the pad, rolling it some distance from the dung source before
burying it. The majority of species of dung beetles found in southern Africa are
crepuscular paracoprids (87%), although the diurnal telecoprids are also numerous,
even if represented by fewer species (12%) (Ferreira, 1972). The telecoprid genus
Kheper is confined mainly to the hot Lowveld areas of Natal and the Transvaal,
extending into tropical Africa.
Telecoprid behavior is considered to have evolved from competition for dung,
as dung is the meeting place of the sexes and is vital for mating success. Dung
beetles, are attracted to dung and arrive by the hundreds at fresh dung where they
either immediately start feeding or burrow into the dung. Within a few minutes,
they distinguish between the sexes, determine the state of maturity of the opposite
sex, and enlist the cooperation of the selected partner in molding and rolling a
brood-ball. If the male has been unsuccessful in finding a female in or on the dung
pad, he might fly away and start secreting a sex attractant or start constructing a
dung ball on his own. The male K. nigroaeneus first buries a brood-ball before
assuming the typical secretion-producing stance at the entrance to the burrow in
which the ball has been buried, or even when he is not in possession of a dung
ball (Edwards and Aschenborn, 1988). Supporting his body on the front two pairs
of legs, the male raises his abdomen and lowers his head so that his body is at
a relatively steep angle. Repeated rapid extension of the hind legs results in the
extrusion of a white proteinaceous material from tiny openings in a depression
on either side of the first abdominal sternite. The secretion is supplied from a
glandular complex underlying the depressions. The movement of the hind legs
results in the secretion being broken up into tiny particles between brushes on the
tibia and comblike structures on the abdomen of the male. Small puffs of secretion
are, thus, released into the air. Amputation of one of the hind legs of a beetle
results in thin-strand fibers resembling cotton wool being formed on that side of
its abdomen. The tibial brushes are rudimentary in the females. When the presence
of an attracted female is detected by a calling male, he challenges the female by
raising his forelegs and advancing on her. Her reaction is a submissive lowering of
the forelegs, followed by antennal contact and acceptance by the male. If the male
has not already prepared and, in the case of K. nigroaeneus, buried a dung ball, the
male detaches a portion of dung from the pad and proceeds to roll both the dung
and the female clinging to it and compacting the dung, away from the dung pad.

SCARABAEINAE SEMIOCHEMICALS VI
503
Once the dung ball is buried, copulation occurs below the soil, the male returning
to the surface after a few days, while the female remains below to lay an egg and
to brood the developing larva (Tribe, 1975, 1976).
The abdominal secretions produced by Kheper species consist of a visible
proteinaceous material that acts as a pheromone-disseminating carrier material
for minute quantities of a complex mixture of volatile organic compounds. A
comparison of the secretions of K. lamarcki, K. nigroaeneus, and K. subaeneus
revealed that similar, but not identical, proteins with molecular masses of ca.
15 kDa are the major constituents of the proteinaceous fractions of each of these
secretions (Burger et al., 1990). The major volatile constituents of the secretion of
K. lamarcki were found to be hexadecanoic acid, 2,6-dimethyl-5-heptenoic acid,
and (E)-nerolidol (Burger et al., 1983). The volatile fractions of the secretions of
these Kheper species are, in fact, complex mixtures, each containing at least 150
constituents such as, among others, hydrocarbons, straight- and branched-chain
fatty acids and methyl and ethyl esters of these, and other fatty acids. The volatile
compoundspresentinthesecretionsofthesethreespeciesaresimilar, butonlyafew
are common to two or all three of the species. The secretions of these insects contain
many chiral long-chain compounds. Because the enantiomers of these compounds
cannot be separated with the current technology, it was decided to suspend attempts
to identify all the volatile constituents of the secretions in favor of the identification
of the EAD-active constituents. In this paper, identification of two EAD-active
constituents of the abdominal secretion of male K. nigroaeneus is presented.
METHODS AND MATERIALS
Collection and Purification of Carrier Material. The pentane, dichlorome-
thane, and chloroform used for extraction purposes were of residue analysis grade
(
5
Merck). All Pyrex glassware used in the handling of the material was heated to
00 C in an annealing oven to remove any traces of organic material. Spatulas,
�
syringes, etc., used in handling the secretion, were cleaned by rinsing with the
dichloromethane specified above.
Adult K. nigroaeneus dung beetles were trapped in Mkuzi Game Reserve in
Kwazulu Natal, South Africa, during the early summer months by baiting pitfall
traps (Tribe, 1976) with fresh horse dung. On arrival in Stellenbosch, one hind
leg was removed from all male beetles. A small percentage of the beetles died
within a week or two after removal of a leg. This was, however, the best method
to prevent dispersal of the secretion that, if not brushed off, collects at one side of
the abdomen. The male and female beetles were kept at subtropical temperatures
in greenhouses, the floors of which were covered with about 15 cm of moist sandy
soil. Permits were granted for the collection and removal from the game reserve of
150 males and a small number of females annually for the duration of this project.

5
04
BURGER AND PETERSEN
When disturbed, male dung beetles immediately stop secreting the attractant
to investigate, and they may even disappear underground together with a female.
Males observed to assume the attractant secreting posture were, therefore, fenced
off from other beetles with aluminum strips. In some cases, the production of se-
cretion continued for up to 2 hr. Without touching the body of the secreting male,
the secreted material was removed periodically from its incapacitated side with
ophthalmic forceps to prevent contamination with dust particles and the evapora-
tion of the pheromone from the carrier material. In the meantime, the vial with
material already collected was kept cool in an icebox. Tiny dust and dung particles
picked up from the abdomen of the insects by the emerging secretion were care-
fully removed from the secretion under a microscope. Only about 5–10% of the
males produced secretion in captivity, and in most instances, secretion production
lasted only a short while and yielded too little material for gas chromatographic
analysis of the secretion from individual insects. Small samples, therefore, had
to be pooled for analysis. However, when secretion production was kept up for
longer periods, it was possible to analyze the secretion from individuals. In a
typical sample preparation, 3 mg of the secretion was sonicated for 1 min with
50 �l of chloroform in a Reacti-Vial, after which the suspension was centrifuged
at 3000 rpm for 10 min. The white carrier material was concentrated in the upper
layers of the solvent, while the remaining dust particles were precipitated. The
solvent, containing soluble organic material, was carefully removed from between
these two layers of solid material with a 100-�l syringe. The process was repeated
a number of times, and extracts were pooled and concentrated for analysis of the
volatile components of the secretion.
Analytical Methods. Gas chromatographic determinations were carried out
with a Carlo Erba 4160 gas chromatograph with parallel flame ionization and
electroantennographic detection (FID/EAD). The instrument was equipped with a
4
0 m � 0.3 mm glass capillary column coated with OV-1701-OH at a film thick-
ness of 0.4 �m. Helium was used as carrier gas at a linear velocity of 28.5 cm/sec at
4
� � � �
0 C, and a temperature program of 2 C/min from 40 C to 250 C was employed.
Chiralgaschromatographicanalysesweredoneonaglasscolumn(25m� 0.3mm)
coated with 10% heptakis(2,3,6-tri-O-methyl)-�-cyclodextrin in OV-1701-OH at
a film thickness of 0.25 �m, using H2 as carrier gas at a linear velocity of 50 cm/sec.
The instrumentation for gas chromatographic analysis of dung beetle secretions
with FID and EAD recording in parallel, with the use of dung beetle antennae as
sensing elements has been described by Burger and Petersen (1991). In short, an
antenna was inserted lengthwise into the air duct, as this arrangement produced
a less noisy baseline than positioning the antenna sideways in the airstream, a
position in which the club of the antenna is subjected to the effect of the turbulent
mixing of the effluent and ambient air. The antenna was protected against heat
�
radiation from the gas chromatograph by a heat shield cooled with water at 18 C.
ElectricalresponsesintheantennawererecordedviaAg–AgClelectrodesplacedin

SCARABAEINAE SEMIOCHEMICALS VI
505
pipets filled with saline solution containing NaCl (7.5 g/liter), CaCl2 (0.21 g/liter),
KCl (0.35 g/liter), and NaHCO3 (0.2 g/liter), as well as polyvinylpyrrolidone
K90 (Fluka, 49 g/liter) to increase its viscosity (Van der Pers, 1980). Antennal
responses were amplified with a Murphy Developments AMS-025 amplifier and
were recorded on an Omniscribe recorder.
Mass spectra were recorded on a Carlo Erba QMD 1000 quadrupole mass
spectrometer with the column and gas chromatographic conditions specified above.
1
13
H and C NMR spectra of synthetic compounds and intermediates were recorded
�
at 299.905 MHz and 75.42 MHz, respectively, at 25 C on a Varian VXR 300 NMR
spectrometer.
3-Methylheptanoic acid was isolated from extracts of the abdominal secretion
by preparative gas chromatography on a glass column (40 m � 0.6 mm) coated
with the apolar phase PS-255 at a film thickness of 1.0 �m. A 1:10 effluent splitter
1
was used, and fractions were collected in CDCl3 for NMR analysis. H NMR
2
3
(
(
1
CDCl3): � = 2.357 (dd, 1H, CHH-2, J2a,2b = 15.0 Hz, J2a,3 = 5.9 Hz); 2.153
2 3
dd,1H, CHH-2, J2b,2a = 15.0 Hz, J2b,3 = 8.0 Hz); 1.96 (m, 1H, CH-3); 1.45–
3
.15 (m, 6H, CH2-4,5,6); 0.894 (t, 3H, CH3-7, J7,6 = 6,7 Hz); 0.970 (d, 3H,
3
CH3-8, J8,3 = 6.6 Hz).
Reference Compounds. 3-Methylindole and starting compounds were pur-
chased from Sigma-Aldrich (Cape Town, South Africa).
Racemic 3-methylheptanoic acid (2) was synthesized from diethyl malonate
according to Scheme 1. Diethyl malonate (1) (23.22 g; 145 mmol) was deproto-
nated with a solution of sodium ethoxide prepared from sodium (3.22 g; 140 mmol)
and absolute ethanol (100 ml), and then alkylated with 2-bromohexane (23.11 g;
140 mmol) (Vogel, 1978). The resulting diethyl ester was saponified by refluxing
it with a solution of potassium hydroxide (20 g) in water (30 ml), after which the
reaction mixture was acidified and refluxed until the evolution of carbon dioxide
had ceased. The conventional isolation procedures gave rac-3-methylheptanoic
1
acid (2) as a colorless oil (12.94 g; 64%); H NMR (CDCl3): � = 2.356 (dd,
H, CHH-2, 2J2a,2b = 14.9 Hz, J2a,3 = 6.0 Hz); 2.145 (dd, 1H, CHH-2,
3
1
2
3
J2b,2a = 14.9 Hz, J2b,3 = 8.1 Hz); 1.96 (m, 1H, CH-3); 1.44–1.14 (m, 6H,
SCHEME 1. Synthesis of racemic 3-heptadecanoic acid.

5
06
BURGER AND PETERSEN
SCHEME 2. Synthesis of (R)-(+)-3-methylheptanoic acid.
3
3
CH2-4,5,6); 0.894 (t, 3H, CH3-7, J7,6 = 6.6 Hz); 0.969 (d, 3H, CH3-8, J8,3 =
6
.6 Hz).
(
R)-(+)-3-Methylheptanoic acid (2a) was synthesized from (R)-(+)-3,
7
-dimethyl-6-octen-1-ol [(R)-(+)-�-citronellol] according to Scheme 2. Isobutene
was slowly introduced into a suspension of Amberlyst H-15 (2.88 g) in a solution
1
9
of (R)-(+)-3,7-dimethyl-6-octen-1-ol (3a) [9.91 g; 63.5 mmol, 98% pure, [�]
+
D
�
5
1
9
3
6
.3 (neat)] in hexane (100 ml) at room temperature for 12 hr (Alexakis et al.,
988). Removal of the catalyst and solvent gave a colorless oil (13.47 g) containing
2
5
9.9% (GC-MS) of (R)-(+)-8-tert-butoxy-2,6-dimethyl-2-octene (4a); [�]
+
D
�
.26 (neat). MS: m/z(%) = 156(6), 138(4), 137(5), 123(6), 109(7), 95(28), 81(67),
1
9(97), 57(100), 55(48), 43(35), 41(94). H NMR (CDCl3): � = 1.599 (d, 3H,
4
3
4
4
CH3-1, J1,3 = 1.4 Hz); 5.102 (m, 1H, == CH-3, J3,4 = 7.1 Hz, J1,3 = J3,9 =
1
.4 Hz); 1.980 (m, 2H, CH2-4); 1.46–1.21 (m, 2H, CH2-5); 1.62–1.47 (m, 1H,
CH-6); 1.62–1.47 (m, 1H, CHH-7 ); 1.46–1.21 (m, 1H, CHH-7); 3.33 (m, 1H,

SCARABAEINAE SEMIOCHEMICALS VI
507
4
CHH-8);3.38(m, 1H, CHH-8);1.679(d, 3H, CH3-9, J9,3 = 1.4Hz);0.890(d, 3H,
3
13
CH3-10, J10,6 = 6.5 Hz); 1.185 (s, 9H, (CH3)3). C NMR (CDCl3): � = 25.72
(
(
(
q, C-1), 131.01 (s, C-2), 124.98 (d, C-3), 25.51 (t, C-4), 37.30 (t, C-5), 29.66
d, C-6), 37.77 (t, C-7), 59.80 (t, C-8), 17.62 (q, CH3-9), 19.72 (q, CH3-10), 72.51
s, C-1 ), 27.62 (q, 3CH3).
0
Ozone was passed through a solution of the unsaturated ether (4a) (13.42 g;
63.3 mmol) in pentane (100 ml) until a sharp color change from colorless to blue
was observed (Marvel and Nichols, 1941). Reduction of the ozonide with lithium
�
aluminum hydride at –10 C, the usual work-up, and column chromatography on
silica gel gave (R)-(+)-6-tert-butoxy-4-methyl-1-hexanol (5a) (7.02 g, 59%) in
5
�
9
1
9.2% purity (GC-MS); [�]2 + 1.95 (neat). MS: m/z(%) = 173(2), 132(6),
D
1
31(15), 115(28), 97(49), 69(40), 59(38), 57(100), 55(78), 43(23), 41(55). H
3
NMR (CDCl3); � = 3.627 (t, 2H, OCH2-1, J1,2 = 6.6 Hz); 1.72–1.47 (m, 2H,
CH2-2); 1.47–1.26 (m, 2H, CH2-3); 1.72–1.47 (m, 1H, CH-4); 1.72–1.47 (m, 1H,
2
CHH-5); 1.47–1.26 (m, 1H, CHH-5); 3.382 (qd, 1H, CHH-6, J6a,6b = 14.7 Hz,
3
3
1
3
2
J6a,5a = 8.6 Hz, J6a,5b = 6.7 Hz); 3.360 (qd, 1H, CHH-6, J6b,6a = 14.6 Hz,
3
J6b,5a = 8.6 Hz, J6b,5b = 7.0 Hz); 0.938 (d, 3H, CH3-7); 1.185 [s, 9H, (CH3)3].
3
C NMR (CDCl3): � = 63.37 (t, C-1), 30.20 (t, C-2), 33.05 (t, C-3), 29.72 (d, C-4),
0
3
7.67 (t, C-5), 59.53 (t, C-6), 19.72 (q, CH3-7), 72.52 (s, C-1 ), 27.61 (q, 3CH3).
Bromination of the alcohol (5a) (6.80 g; 36.20 mmol) with tetrabromome-
thane (14.37 g; 43.33 mmol) and triphenylphosphine (10.42 g; 39.72 mmol;)
in acetonitrile (40 ml) (Christol et al., 1978; Slagle et al., 1981) gave (R)-(+)-
1
-bromo-6-tert-butoxy-4-methylhexane (6a) as a colorless oil (8.74 g, 96.4%)
25
�
in 86% purity (GC-MS); [�] + 3.46 (neat). MS: m/z (%) = 237(3), 235(3),
D
1
9
79(9), 177(10), 150(2), 148(2), 137(9), 135(10), 123(6), 121(7), 109(6), 107(7),
7(92), 69(85), 59(88), 57(100), 56(77), 55(92), 43(86), 41(95). HNMR(CDCl3):
1
3
�
= 3.354 (t, 2H, CH2-1, J1,2 = 7.0 Hz); 1.98–1.76 (m, 2H, CH2-2); 1.49–1.21
(m, 2H, CH2-3); 1.72–1.42 (m, 1H, CH-4); 1.69–1.50 (m, 2H, CH2-5); 3.396
2
3
3
(
(
3
qd, 1H, CHH-6, J6a,6b = 14.3 Hz, J6a,5a = 8.9 Hz, J6a,5b = 6.9 Hz); 3.354
2 3 3
qd, 1H, CHH-6, J6b,6a = 14.9 Hz, J6b,5a = 8.8 Hz, J6b,5b = 6.9 Hz); 0.900 (d,
3
13
H, CH3-7, J7,4 = 6.6 Hz); 1.85 [s, 9H, (CH3)3]. C NMR (CDCl3): � = 34.29
(t, C-1), 30,41 (t, C-2), 35.57 (t, C-3), 29.35 (d, C-4), 37.87 (t, C-5), 59.53 (t, C-6),
0
1
9.72 (q, CH3-7), 72.49 (s, C-1 ), 27.62 (q, 3CH3).
Treatment of the bromide (6a) (7.35 g; 29.3 mmol) with lithium dimethyl-
�
cuprate in ether at 0 C (Johnson and Dutra, 1973) and isolation of the prod-
uct in the usual manner gave (R)-1-tert-butoxy-3-methylheptane (7a) as a color-
less oil (4.04 g, 74%) in 85% purity (GC-MS). MS: m/z (%) = 171(2), 113(3),
1
8
(
6
6
6
4(3), 71(28), 69(7), 59(25), 57(100), 56(18), 55(17), 43(35), 41(43). H NMR
2
3
3
CDCl3): � = 3.370 (qd, 1H, CHH-1, J1a,1b = 14.9 Hz, J1a,2a = 8.8 Hz, J1a,2b =
2
3
3
.0 Hz); 3.344 (qd, 1H, CHH-1, J1b,1a = 14.9 Hz, J1b,2a = 8.7 Hz, J1b,2b =
.5 Hz); 1.62–1.46 (m, 2H, CH2-2); 1.70–1.46 (m, 2H, CH-3); 1.46–1.05 (m,
3
H, CH2-4,5,6); 0.899 (t, 3H, CH3-7, J7,6 = 6.6 Hz); 0.871 (d, 3H, CH3-8,

5
08
BURGER AND PETERSEN
3
13
J8,3 = 6.6 Hz); 1.185 [s, 9 H, (CH3)3]. C NMR (CDCl3): � = 59.88 (t, C-1),
3
7.87 (t, C-2), 25.65 (d, C-3), 36.88 (t, C-4), 29.21 (t, C-5), 23.01 (t, C-6), 14.17
0
(q, C-7), 19.78 (q, CH3-8), 72.42 (s, C-1 ), 27. 63 (q, 3CH3).
Treatment of the tert-butoxy ether (7a) (3.21 g; 17.2 mmol) with iodotri-
methylsilane (6.18 g; 30.91 mmol) in dichloromethane (50 ml) (Jung and Lyster,
977) gave (R)-3-methyl-1-heptanol (8a) (2.24 g) in quantitative yield and 86%
1
purity (GC-MS). MS: m/z(%) = 112(2), 97(3), 84(52), 83(25), 70(77), 69(37),
1
5
(
7(38), 56(67), 55(98), 43(100), 44(38), 41(87). H NMR (CDCl3): � = 3.654
3
t,2H, OCH2-1, J1,2 = 6.6 Hz); 1.66–1.48 (m,2H, CH2-2); 1.69–1.48 (m,1H,
3
CH-3); 1.47–1.11 (m,6H, CH2-4,5,6); 0.896 (t, 3H, CH3-7, J7,6 = 6.7 Hz); 0.894
(
(
(
d, 3H, CH3-8). 13C NMR (CDCl3): � = 61.28 (t, C-1), 40.01 (t, C-2), 27.35
d, C-3), 36.82 (t, C-4), 29.20 (t, C-5), 22. 97 (t, C-6), 14.12 (q, C-7), 19.66
q, CH3-8).
Oxidation of the alcohol (8a) with various reagents such as chromic acid
and permanganate gave the acid (2a) in very poor yields. The best results were
obtained with calcium hypochlorite as oxidizing agent (Kabalka et al., 1990).
Concentrated hydrochloric acid (7 ml) was, thus, added dropwise to a stirred
suspension of hypochlorite (8.8 g; 61.5 mmol) in water (40 ml) giving a bright
yellow solution. A mixture of tert-butanol (30 ml) and tetrachloromethane (20 ml)
was added, followed by the addition of (R)-3-methyl-1-heptanol (8a) (1.87 g;
14.4 mmol) in tert-butanol (10 ml) over a period of 4 min. The reaction mixture
was stirred for 5 hr, and the organic material extracted exhaustively with tetra-
chloromethane. The combined extracts were washed with a solution of sodium
metabisulfite until it was colorless. The organic acid was extracted with sodium
carbonate, and this basic solution was extracted thoroughly with ether to re-
move all non-acidic products. The water layer was carefully acidified with hy-
drochloric acid and the organic acid extracted with ether. The usual work-up
procedures gave (R)-(+)-3-methylheptanoic acid (2a) as a colorless oil (1.14 g,
5
�
5
5%) in 99.3% purity (GC-MS); [�]2 + 4.94 (neat); 96.2% ee by gas chromatog-
D
raphy on heptakis(2,3,6-tri-O-methyl)-�-cyclodextrin. MS: m/z (%) = 144(3),
1
6
2
2
29(8), 115(3), 101(7), 87(55), 85(28), 84(33), 73(20), 69(22), 61(26),
1
0(100), 56(20), 55(38), 45(27), 43(75), 42(26), 41(65). H NMR (CDCl3): � =
2
3
.355 (dd,1H, CHH-2, J2a,2b = 14.9 Hz, J2a,3 = 5.9 Hz); 2.144 (dd, 1H, CHH-
2
3
, J2b,2a, = 14.9 Hz, J2b,3 = 8.1 Hz); 1.96 (m, 1H, CH-3); 1.46–1.11 (m, 6H,
3
CH2-4,5,6); 0.894 (t, 3H, CH3-7, J7,6 = 6.7 Hz); 0.968 (d, 3H, CH3-8,
3
13
J8,3 = 6.6 Hz). C NMR (CDCl3): � = 190.40 (s, C-1), 41.56 (t, C-2), 30.16
(
d, C-3), 36.37 (t, C-4), 29.11 (t, C-5), 22.78 (t, C-6), 14.07 (q, C-7), 19.72
(q, CH3-8).
(
S)-(� )-3-methylheptanoic acid was synthesized in an analogous manner
from (S)-(� )-�-citronellol in 51% yield, 99.2% purity (GC-MS), and 96.8% ee;
MS: m/z(%) = 144(3), 129(9), 115(10), 101(7), 87(55), 85(38), 84(33), 73(20),
6
9(21), 61(25), 60(100), 56(19), 55(36), 45(26), 43(73), 42(25), 41(65), 39(30).

SCARABAEINAE SEMIOCHEMICALS VI
509
RESULTS AND DISCUSSION
A typical FID gas chromatogram of an extract of the abdominal secretion of
the male K. nigroaeneus is shown in Figure 1. A faster programming rate and the
injection of more material were found to produce a better signal-to-noise ratio in
the EAD recording. Dung beetle antennae survived in EAD experiments for more
than 12 hours. Figure 2 shows the first part of a gas chromatographic analysis with
�
EAD and FID recording in parallel at a programming rate of 4 C/min. A female
antenna was used as the EAD-sensing element in this analysis. Although male and
female antennae mostly produced reproducible and qualitatively similar results, a
few examples of male and female antennae responding reproducibly to a few other
long-chain constituents of the secretion were encountered over a period of several
years. Because these EAD analyses could not be reproduced with other antennae,
identification of these compounds could not be verified by EAD analyses of the
synthetic compounds. However, they were always long-chain fatty acids such
as hexadecanoic acid and/or the methyl or ethyl esters of long-chain acids. It is
difficult to explain these results, and this phenomenon was not investigated in more
detail.
One explanation for the presence of a large number of long-chain alkanes,
alcohols, fatty acids, etc., in the abdominal secretion could be that these com-
pounds augment the pheromone disseminating capacity of the proteinaceous car-
rier material. However, the fact that the antennae of the male and female dung
beetles on occasion seemed to respond to some of these compounds could indicate
that, depending on circumstances, they possibly have a semiochemical function.
FIG. 1. Total ion chromatogram (GC-MS) of an extract of the male abdominal secretion of
the dung beetle Kheper nigroaeneus. Gas chromatographic conditions: 40 m � 0.3 mm-ID
glass column coated with OV-1701-OH at a film thickness of 0.375 �m and programmed
�
�
�
�
�
at 2 /min from 40 to 250 C (hold); injector temperature 220 C; FID 250 C.

5
10
BURGER AND PETERSEN
FIG. 2. First part of a gas chromatogram of an extract of the male abdominal secretion of the
dung beetle Kheper nigroaeneus obtained by EAD/FID recording in parallel using a female
antenna as detector element. Corresponding peaks in the EAD and FID chromatograms are
indicated with a dot. Gas chromatographic conditions as in Figure 1 but with a programming
�
�
�
�
�
rate of 4 C/min from 40 to 250 C (hold); EAD interface line 250 C; antenna and humidified
air at 22 C. Assignment of EAD-active constituents: A = unidentified; B = (R)-(+)-3-
methylheptanoic acid; C = skatole.
Although some of these long-chain compounds are common to the four Kheper
species that were investigated, it is noteworthy that there were clearly large qual-
itative and quantitative differences in their secretions. This could be construed as
evidence that the volatile constituents of the secretions are involved in the trans-
mission of chemical signals. It is also possible, however, that the EAD and gas
chromatographic techniques used in this study were not suitable for the detection
of such long-chain compounds.

SCARABAEINAE SEMIOCHEMICALS VI
511
Constituent A, eluting at 18.55 min in the gas chromatographic analysis of
the volatile organic fraction of the abdominal secretion shown in Figure 2, gave
reproducible EAD responses, but was invisible in FID gas chromatograms despite
relatively large samples having been used for the analysis. The low concentration in
which this compound is present in the secretion and the fact that it was impossible
to collect large enough quantities of abdominal secretion so far has precluded its
identification.
Constituent B, eluting at 20.92 min, was initially identified as 5-methylhepta-
noic acid. However, it was found that the retention time and mass spectra of
synthetic 5-methylheptanoic acid are different from those of the natural com-
pound. 5-Methylheptanoic acid, furthermore, did not elicit any EAD response in
the antennae of male or female K. nigroaeneus. All the available abdominal secre-
tions and extracts of the secretion accumulated over more than eight years were
combined for preparative gas chromatography, and enough material was isolated
1
for H NMR analysis of this acid, which was identified as 3-methylheptanoic acid.
Racemic 3-methylheptanoic acid (2) was synthesized from diethyl malonate
(
1) and 2-bromohexane according to Scheme 1. (R)-(+)-3-Methylheptanoic
acid (2a) was synthesized from (R)-(+)-3,7-dimethyl-6-octen-1-ol [(R)-(+)-�-
1
citronellol] (3a) according to Scheme 2 and was found to have mass and H NMR
spectra identical to those of the natural compound. This synthetic enantiomer
was found to be EAD active. The S enantiomer was synthesized from (S)-(� )-�-
citronellol according to an analogous synthetic route and was found to be EAD
inactive. The two enantiomers are separable on a capillary column coated with a
mixture of the stationary phase OV-1701-OH and heptakis (2,3,6-tri-O-methyl)-
�
-cyclodextrin, the S enantiomer eluting first from this column. Finally, a gas
chromatographic analysis with FID and EAD detection in parallel and using this
chiral column confirmed the EAD activity of the second eluting R enantiomer.
The third EAD-active constituent of the secretion, C, eluting at 29.21 min
in the gas chromatogram shown in Figure 2, was identified as 3-methylindole
(skatole), which has also been identified as a constituent of the abdominal secretion
of K. lamarcki. Synthetic 3-methylindole was found to be EAD active.
One of the goals of this research was the development of a pheromone-
based monitoring system for some of the Kheper species as a possible method to
evaluate certain aspects of the ecological status of areas where these dung beetles
are found. So far, all attempts at devising a field test with which the constituents
of the secretion could be evaluated have been unsuccessful. The primary source of
attraction is dung, and in tests with constituents of the secretion of K. lamarcki, for
example, no dung beetles were attracted to pitfall traps baited with hexadecanoic
acid, 2,6-dimethyl-5-heptenoic acid, (E)-nerolidol, and skatole. In the case of
K. lamarcki, initial indications that more females than males are attracted to pitfall
traps baited with dung plus these compounds than to traps baited only with dung
proved to be statistically insignificant. In this regard, however, it must also be taken

5
12
BURGER AND PETERSEN
into consideration that the ratio of males to females constantly changes due to the
fact that the females stay underground for a while after mating. The sex ratio could
also be influenced by other factors, such as the localized availability of dung and
differences in the texture and moisture content of the soil in different parts of the
area where the tests were done.
Acknowledgments—Support of this work by the University of Stellenbosch and the Foundation
for Research Development, Pretoria, is gratefully acknowledged. The authors are indebted to the Natal
Parks, Game, and Fish Preservation Board for permission to study dung beetles in Mkuzi Game Reserve.
We thank Dr. H. Geertsema, Department of Entomology and Nematology, University of Stellenbosch,
for reading the manuscript.
REFERENCES
ALEXAKIS, A., GARDETTE, M., and COLIN, S. 1988. Mild protection and deprotection of alcohols as
ter-butyl ethers in the field of pheromone synthesis. Tetrahedron Lett. 29:2951–2954.
BORNEMISSZA, G. F. 1969. A new type of brood care observed in the dung beetle Oniticellus cinctus
(Scarabaeidae). Pedobiologia 9:223–225.
BURGER, B. V. and PETERSEN, W. G. B. 1991. Semiochemicals of the Scarabaeinae III: Identification
of the attractant for the dung beetle Pachylomerus femoralis in the fruit of the spineless monkey
orange tree, Strychnos madagascariensis. Z. Naturforsch. 46c:1073–1079.
BURGER, B. V., MUNRO, Z., RO¨TH, M., SPIES, H. S. C., TRUTER,V., TRIBE, G. D., and CREWE, R. M.
1
983. Composition of the heterogeneous sex attracting secretion of the dung beetle, Kheper
lamarcki. Z. Naturforsch. 38c:848–855.
BURGER, B. V., MUNRO, Z. M., and BRANDT, W. F. 1990. Pheromones of the Scarabaeinae II: Compo-
sition of the pheromone disseminating carrier material secreted by male dung beetles of the genus
Kheper. Z. Naturforsch. 45c:863–872.
CHRISTOL, S. J., STROM, R. M., and STULL, D. P. 1978. Bridged polycyclic compounds. 86. Multi-
ple mechanisms in the reactions of some bridged alcohols with triphenylphosphine and carbon
tetrachloride. J. Org. Chem. 43:1150–1154.
EDWARDS, P. B. and ASCHENBORN, H. H. 1988. Male reproductive behaviour of the African ball-rolling
dung beetle, Kheper nigroaeneus (Coleoptera: Scarabaeidae). Coleopt. Bull. 42:17–27.
´
FERREIRA, M. C. 1972. Os Escarabideos de Africa (Sul do S a´ ara). Rev. Entomol. Mocambique 11:5–
088.
1
Halffter, G. and MATTHEWS, E. G. 1966. The natural history of dung beetles of the subfamily
Scarabaeinae (Coleoptera, Scarabaeidae). Folia Entomol. Mex. 12/14:1–32.
HEINRICH, B. and BARTHOLOMEW, G. A. 1979. The ecology of the African dung beetle. Sci. Am.
2
41:118–126.
HUGHES, R. D. 1970. The bushfly. Aust. Nat. Hist. 16:331–334.
JOHNSON, C. R. and DUTRA, G. A. 1973. Reactions of lithium diorganocuprates(I) with tosylates.
Synthetic aspects. J. Am. Chem. Soc. 95:7777–7782.
JUNG, M. E. and LYSTER, M. A. 1977. Quantitative dealkylation of alkyl ethers via treatment with
trimethylsilyl iodide. A new method for ether hydrolysis. J. Org. Chem. 42:3761–3764.
KABALKA, G. W., CHATLA, N., WADGAONKAR, P. P., and DESHPANDE, S. M. 1990. The conversion
of aliphatic primary alcohols to the corresponding carboxylic acids using calcium hypochlorite.
Synth. Commun. 20:1617–1623.
MARVEL, C. S. and NICHOLS, V. 1941. The formation of dimeric peroxides by ozonizations of olefins
(1). J. Org. Chem. 6:296–304.

SCARABAEINAE SEMIOCHEMICALS VI
513
SLAGLE, J. D., HUANG, T. T. S., and FRANZUS, B. 1981. Mechanisms of the triphenylphosphine-
tetrachloromethane-alcohol reaction. Pericyclic or clustered ion pairs? J. Org. Chem. 46:3526–
3
530.
TRIBE, G. D. 1975. Pheromone release by dung beetles (Coleoptera: Scarabaeidae). S. Afr. J. Sci. 71:
77–278.
2
TRIBE, G. D. 1976. The ecology and ethology of ball-rolling dung beetles (Coleoptera: Scarabaeidae).
MSc thesis. University of Natal, Pietermaritzburg, South Africa, pp. 35–36.
VAN DER PERS, J. N. C. 1980. Olfactory receptors in small ermine moths (Lepidoptera: Yponomeuti-
dae): Electrophysiology and morphology. Ph.D. thesis. University of Groningen, p. 39.
VOGEL, A. I. 1978. Textbook of Practical Organic Chemistry, 4th ed. Longman, New York, p. 491.
WATERHOUSE, D. F. 1974. The biological control of dung. Sci. Am. 230:100–109.