RESEARCH PAPER

Chemical composition and pheromonal functionof the defensive secretions in the subtribe Stizopina(Coleptera, Tenebrionidae, Opatrini)

S. Geiselhardt Æ T. Schmitt Æ K. Peschke

Received: 13 March 2008 / Accepted: 25 November 2008 / Published online: 15 January 2009

� Birkhauser Verlag, Basel/Switzerland 2009

Abstract The chemical composition of the defensive

secretions of 52 species from 15 genera of the tenebrionid

subtribe Stizopina was analyzed. The secretions of all

species contained 1,4-benzoquinones, 1-alkenes, and

monoterpene hydrocarbons, only one species was lacking

the latter. Methyl- and ethyl-1,4-benzoquinone were

ubiquitous, mostly accompanied by smaller amounts of

1,4-benzoquinone as well as isopropyl- and propyl-1,4-

benzoquinone. 1-Alkenes were dominated by 1-undecene

with varying admixtures of other 1-alkenes. The mono-

terpene hydrocarbons always consisted of a mixture of

a-pinene, camphene, b-pinene and limonene, but also

p-cymene, a-terpinene or a-phellandrene were found in

some species. Furthermore, some species synthesized

additional compounds such as phenols, ketones, 2,5-dihy-

droxy-6-methylbenzoate, 2-hydroxy-4-methoxyacetophe-

none and naphthoquinones. Bioassays showed that the

defensive secretion co-functioned as an aggregation pher-

omone in the subtribe Stizopina. All nine tested species

from six genera were attracted to defensive secretion of

Stizopina species, but they did not distinguish between

defensive secretions of different Stizopina species. This

lack of discrimination might be the initial step for the

formation of interspecific aggregations and the evolution

of cleptoparasitism within the Stizopina.

Keywords Defensive secretion � Aggregation

pheromone � Benzoquinones �Monoterpene hydrocarbons �1-Alkenes � Coleoptera � Tenebrionidae

Introduction

The chemical defensive system of Tenebrionidae is one of

the best studied among insects. More than 200 species of

110 genera and 33 tribes have hitherto been analyzed

(Brown et al. 1992; Gnanasunderam et al. 1981; Howard

1987; Tschinkel 1975a). The secretions are primarily made

up of quinones. Methyl- and ethyl-1,4-benzoquinone are

ubiquitous, mostly accompanied by unsaturated hydrocar-

bons, with varying chain lengths. Besides these two major

classes several compounds belonging to other chemical

classes are found in minor amounts. These are monoter-

pene hydrocarbons (Brown et al. 1992; Geiselhardt et al.

2006a; Gnanasunderam et al. 1981), ketones (Gnanasun-

deram et al. 1985; Peschke and Eisner 1987; Tschinkel

1975b), and phenols (Attygalle et al. 1991; Brown et al.

1992; Lloyd et al. 1978; Tschinkel 1969).

Tenebrionids of the subtribe Stizopina (tribe Opatrini)

are all gregarious, nocturnal detritivores that mostly inhabit

the arid regions of Namibia and western South Africa (Koch

1963). The defensive secretions of two species, Parastiz-

opus armaticeps and Eremostibes opacus, have been

analyzed so far (Geiselhardt et al. 2006a). Both species

contain 1,4-benzoquinones, 1-alkenes, and monoterpene

hydrocarbons in their defensive secretion.

The subsocial P. armaticeps shows biparental brood

care with division of labor and raises its brood in self dug

burrows, which the beetles subsequently use as shelters

throughout the year (Rasa 1990). In the Kalahari Desert,

P. armaticeps is always found associated with E. opacus

S. Geiselhardt � T. Schmitt � K. Peschke

Institut fur Biologie I, Universitat Freiburg,

Hauptstraße 1, 79104 Freiburg, Germany

S. Geiselhardt (&)

Institut fur Biologie, Haderslebener Straße 9,

12163 Berlin, Germany

e-mail: sven.geiselhardt@fu-berlin.de

Chemoecology (2009) 19:1–6

DOI 10.1007/s00049-008-0001-7 CHEMOECOLOGY

that becomes a cleptoparasite during the breeding season of

P. armaticeps (Rasa 1996). Interspecific aggregations are

also found in other Stizopina species and in all cases one of

the partners is a brood caring species (Rasa and Endrody-

Younga 1997). The formation of the aggregations between

P. armaticeps and E. opacus is mediated by volatiles (Rasa

1994). E. opacus is attracted to the scent of its host, but is

unable to discriminate between odor of its host and con-

specifics, whereas odor of other tenebrionids is avoided.

Responsible for the attraction are monoterpene hydrocar-

bons, especially (-)-camphene, originating from the

defensive secretion of P. armaticeps (Geiselhardt et al.

2006a). Both species share the same set of monoterpene

hydrocarbons, but whether E. opacus has ‘broken the code’

of P. armaticeps and mimics its host, or whether the

association is based on odor homology, and defensive

secretions function as aggregation pheromones in all Sti-

zopina, is still speculative.

In the present study, we analyzed and compared the

chemical compositions of the defensive secretions of 52

species from 15 genera of the subtribe Stizopina to test the

hypothesis of odor homology. Furthermore, in choice

experiments with nine species from six genera we tested, if

defensive secretion functions as aggregation pheromone in

all these species, and if so, whether these species dis-

criminate between defensive secretion of conspecifics and

that of other Stizopina.

Materials and methods

Beetles

Beetles were collected in South Africa and Namibia during

three collection trips from August to October 2002, May to

June 2003, and September to October 2003. Each species

was kept at 25�C in a separate terrarium, containing sand,

shelters, and a water source. All species were fed with oat

flakes ad libitum.

Collection of defensive secretion

Defensive secretions were milked from beetles by holding

a small piece of filter paper against the junction of the

elytra and the anal sternite. The filter papers were extracted

in 0.5 ml n-pentane (Brown et al. 1992).

Chemical analysis

Defensive secretions were analyzed on a coupled gas

chromatography–mass spectrometer system (HP 6890 ser-

ies GC–HP 5973 MSD) equipped with a split/splitless

injector (300�C) and an autosampler (injection of 1 ll). A

fused silica column (DB-1, 30 m 9 0.25 mm ID, 0.25 lm,

J & W Scientific, Folsom, CA, USA) was used with a

helium flow of 1 ml/min. The oven temperature was pro-

grammed as follows: 2 min at 50�C, to 250�C at 6�C/min, to

300�C at 20�C/min. Electron impact ionization was 70 eV.

Compounds were identified by comparing mass spectra

and retention times with those of authentic standards or

previously identified reference compounds of other teneb-

rionids. Double-bond positions in olefins were determined

by interpreting the mass spectra of the dimethyl disulfide

adducts (Francis and Veland 1981).

Enantioselective analysis of monoterpene hydrocarbons

were performed on a HP 6890 GC fitted with a flame

ionization detector (300�C). Samples (1 ll) were injected

in splitless mode. A b-DEXTM120 fused silica column

(30 m 9 0.25 mm ID 9 0.25 lm, Supelco, Deisendorf,

Germany) was used for enantiomeric separation with a

helium flow of 1 ml/min. The oven was programmed with

2 min at 35�C, then 5�C/min to 270�C (15 min). Enantio-

mers were identified by comparing the retention times with

those of authentic standards [all from Fluka, except (?)-

camphene from Merck] run under identical GC conditions.

Additionally, (?)- or (-)-enantiomers, quantitatively

adjusted to the equivalent of the corresponding monoter-

pene in the sample, were added to the defensive secretions

of Adoryacus bidens, Amathobius mesoleius, Blenosia

namaquensis, Calaharena dutoiti, Eremostibes barbatus,

E. bushmanicus, E. opacus, Ennychiatus caraboides,

Periloma alfkeni, P. a. armaticeps, Planostibes cribricollis,

and Pl. namaqua, respectively.

Choice experiments

Choice experiments were conducted in glass terraria

(50 9 50 9 20 cm) kept in an environmental chamber at

25�C, 40% RH and a 14L:10D photoperiod. Each terrarium

was thinly floored with sand and contained 12 circular

shelters (6 cm OD; 2 cm high), with small entrances,

arranged in a distance of 5 cm from the walls. Two shelters

contained a rubber septum (violett 11 mm, Analyt,

Mullheim, Germany) onto which the defensive secretions

were applied. At the beginning of the scotophase, one

septum was soaked with defensive secretion of the species

to be tested and the other with defensive secretion of P. a.

armaticeps. In the case of P. a. armaticeps, heterospecific

defensive secretion was obtained from E. opacus. The

applied amounts were 100 lg in all cases. After application

of volatiles, 12 beetles were introduced into the center of

each terrarium, and their distribution was registered in the

next photophase. For each species 15 replications were

carried out. The test criterion for the v2 test was whether or

not the largest aggregation was in a shelter with defensive

secretion.

2 S. Geiselhardt et al.

Results

Chemical composition of the defensive secretions

The defensive secretions of all 52 analyzed species from 15

genera comprised 1-alkenes, 1,4-benzoquinones (BQs) and

monoterpene hydrocarbons, together with small amounts of

miscellaneous compounds (Table 1). The sole exception

was Psammogaster malani which secreted no monoterpene

hydrocarbons.

Among the 1,4-BQs, ethyl- and methyl-1,4-BQ were

most abundant, followed by 1,4-BQ. Isopropyl- and propyl-

1,4-BQ were mostly minor compounds, but A. bidens

(11.2%), Microstizopus ciliatus (2.3%) and Namazopus

parallelus (6.1%) showed substantial amounts of isopro-

pyl-1,4-BQ in their secretions. Occasionally, smaller

amounts of the corresponding 1,4-hydroquinones could be

found as well in the defensive secretions.

The 1-alkene fraction was mostly dominated by the

ubiquitous 1-undecene. The presence and abundance of the

other 1-alkenes was highly variable, even within species.

1-Nonadecene and 1-tridecene were found in nearly all

secretions, but only as minor compounds. In contrast,

1-pentadecene was only synthesized by nine species, but in

five of them as predominant 1-alkene. 1-Heptadecene was

present in more than 50% of the secretions and could

contribute up to 25% in some species. Besides these

1-alkenes, some species contained 1,6- and 1,8-pentade-

cadiene and/or 1,6-heptadecadiene, but only in A. bidens

made these compounds up more than 1%.

The monoterpene hydrocarbon fraction always consisted

of a mixture of a-pinene, camphene, b-pinene and limo-

nene. Sabinene was secreted by about half of the species,

but only in traces. Additionally, P. major and P. lithop-

sophilus synthesized p-cymene (both 0.2%), the latter

together with a-phellandrene (1.8%), and secretions of

Pl. cribricollis and Pl. scymnulus contained a-terpinene

(0.5 and 1.7%). In all 12 species of 9 genera, in which we

analyzed the configuration of the monoterpene hydrocar-

bons, only the (-)-enantiomers could be detected.

Some of the species were characterized by the presence

of phenolic compounds. 3-Methylphenol was detectable in

the secretions of 14 species, but only Pl. scymnulus (4.0%)

and Adoryacus metasternalis (1.7%) showed proportions

greater than 1%. 3-Ethylphenol was only produced in

A. metasternalis in noteworthy amounts (4.9%), whereas

phenol was always a trace compound, if present at

all. Among the minor components of other classes of

compounds, ketones were the most frequent ones.

2-Undecanone was nearly ubiquitous, but also traces of

2-tridecanone and 2-pentdecanone were found in Ennyc-

hiatus fitzsimonsi and Ennychiatus namaquanus, as well as

small amounts of 3-tridecanone in Microstizopus mantipes.

In addition, En. namaquanus produced small amounts of

methyl 2,5-dihydroxy-6-methylbenzoate (0.4%) while

M. mantipes contained 2-hydroxy-4-methoxyacetophenone

(1.7%). In some cases, traces of naphthoquinone and its

alkyl derivatives were detectable in the defensive secre-

tions of P. a. armaticeps.

Choice experiments

All nine tested species from six genera of Stizopina were

attracted to defensive secretion (Fig. 1). The largest

aggregations of A. bidens, E. bushmanicus, E. opacus, and

Pl. namaqua were always found in shelters with defensive

secretion (N = 15, v2 = 75.0, P \ 0.001), those of Am.

mesoleius, E. barbatus, and P. a. armaticeps to 93.3%

(N = 15, v2 = 63.5, P \ 0.001), and those of B. namaq-

uensis and Pl. cribricollis to 86.7% (N = 15, v2 = 52.9,

P \ 0.001). Furthermore, none of the species was dis-

criminating between shelters with their own defensive

secretion (A) and those with defensive secretion of another

Stizopina species (B). The number of largest aggregations

was equally distributed among both conditions, with a

mean A/B ratio of 1.09 ± 0.25 (Fig. 1; for all species:

N = 15, v2 \ 1, P [ 0.05).

Discussion

Chemical compositions of the defensive secretions

The composition of the pygidial defensive secretions of the

subtribe Stizopina corresponded to previous findings con-

cerning other tenebrionid beetles, which have shown the

ubiquity of methyl- and ethyl-1,4-BQ, mostly accompanied

by 1-alkenes of varying chain lengths (Brown et al. 1992;

Tschinkel 1975a). In addition to these compound, the

secretions of Stizopina species contained substantial

amounts of monoterpene hydrocarbons. These are very rare

compounds in the defensive secretion of tenebrionids, most

prominent in the tribes Titaenini and Coelometopini

(Brown et al. 1992; Gnanasunderam et al. 1981), although

they are widespread among other beetle families (Francke

and Dettner 2005), as well as other insects orders (Blum

1981). Within the tribe Opatrini to which the Stizopina

belong, Gonocephalum sp. synthesizes a-pinene (Brown

et al. 1992), but other monoterpenes have not been repor-

ted. Thus, the co-occurrence of a-pinene, camphene,

b-pinene and limonene seems to be a specific characteristic

for the subtribe Stizopina. The fact that all analyzed species

possess only the (-)-enantiomers of the monoterpenes,

may suggest to assume that this is the same in all other

Stizopina. The uniformity of the compositions of the

monoterpenes corroborates the hypothesis of odor

Chemical composition and pheromonal function of the defensive secretions in the subtribe Stizopina 3

Table 1 Chemical composition of the pygidial defensive secretions of 52 species from 15 genera of Stizopina. B 1%; = 1–10%; C10%

senepretonoMseicepS Benzoquinones Phenols 1-Alkenes

-Pin

ene

Cam

phen

e

Sabi

nene

-Pin

ene

p-C

ymen

e

-Ter

pine

ne

-Phe

lland

rene

Lim

onen

e

1,4-

Met

hyl-

1,4-

Eth

yl-1

,4-

Isop

ropy

l-1,

4-

Prop

yl-1

,4-

Phen

ol3-

Met

hyl-

3-

Eth

yl-

1-C

9ene

1-C

11en

e

1-C

13en

e

1-C

15en

e

1-C

17en

e

1,6-

/1,8

-C15

dien

e

1,6-

C17

dien

e

othe

rs

Adoryacus bidens Adoryacus metasternalis

Amathobius mesoleius Amathobius subplanatus

Blenosia exarata Blenosia monticola Blenosia namaquensis Blenosia planiuscula Blenosia semicostata Blenosia sulcata

Blacodatus vertagus

Calaharena dutoiti

Ennychiatus caraboides Ennychiatus fitzsimonsi Ennychiatus namaquanus

Eremostibes barbatus Eremostibes bushmanicus Eremostibes clavifemur Eremostibes megatibia Eremostibes opacus

Microstizopus arthridoideus Microstizopus ciliatus Microstizopus femoralis Microstizopus infradentatus Microstizopus mantipes Microstizopus transvaalensis

Namazopus parallelus

Nemanes expansicollis

Parastizopus a. armaticeps TParastizopus a. armaticeps GParastizopus a. bifidus Parastizopus a. coronatus Parastizopus a. occipitalis Parastizopus lithopsophilus Parastizopus major Parastizopus transgariepinus

Periloma alfkeni

Planostibes angulatipes Planostibes byrrhoides Planostibes curvatus Planostibes cribricollis Planostibes dentipes Planostibes hereroensis Planostibes namaqua Planostibes promontorii Planostibes recurvus Planostibes rufipes Planostibes scymnulus

Psammogaster malani

Stizopus mammifer Stizopus propleuralis Stizopus talpa

α α αβ

4 S. Geiselhardt et al.

homology as the basis of interspecific aggregations. The

absence of monoterpenes in Ps. malani is rather a sec-

ondary loss than the primary state, as Ps. malani is thought

to be a highly derived species (Koch 1963; Penrith 1984).

Besides the ubiquitous 1,4-BQs, 1-alkenes and mono-

terpenes, there was a variety of other compounds that were

found in only few species or even a single one. All these

substances have already been reported from other darkling

beetles. Phenols are produced in the pygidial defensive

gland of tenebrionids of different subfamilies (Attygalle

et al. 1991; Brown et al. 1992), as well as in the prothoracic

defensive gland of Zophobas rugipes (Tschinkel 1969).

2-Tridecanone is secreted by Blaps mucronata (Peschke

and Eisner 1987) and 2-pentadecanone by Uloma tenebr-

ionoides, together with 2-heptadecanone and unsaturated

2-ketones (Gnanasunderam et al. 1985). In tenebrionid

beetles, 2-hydroxy-4-methoxyacetophenone and methyl

2,5-dihydroxy-6-methylbenzoate were previously only

known from the defensive secretions of some members of

the flour beetle genus Tribolium (Howard 1987) and

naphthoquinone and its 6-alkyl derivatives from the genera

Argoporis (Tschinkel 1972) and Hypophloeus (Dettner

1993). Some of these compounds were previously thought

to be characteristic for a specific species or species group

within the darkling beetles and were used as chemotaxo-

nomic characters in Tribolium (Howard 1987). The

occurrence of these compounds in the subtribe Stizopina

shows the limitation of defensive compounds as chemo-

taxonomic characters, although the specific mixture of

monoterpenes seems to be typical for the Stizopina.

Defensive secretion as aggregation pheromone

In members of the subtribe Stizopina, the pygidial defen-

sive secretion serves multiple functions. The primary

function of the secretion is to deter predators, but, in

addition, the secretion co-functions as an aggregation

pheromone. Although pheromone-mediated aggregations

are a widespread phenomenon in insects (Wertheim et al.

2005), only in a few cases, defensive secretions have been

adopted to function as aggregation pheromones (Blum

1996). In the tenebrionid genus Blaps, the defensive

secretion also includes this additional functions. High

concentrations of defensive secretion elicit a strong escape

behavior in aggregated Bl. mucronata (Tannert and Hien

1973), whereas low concentrations have the opposite effect

and attract adults of Bl. mucronata (Tannert and Hien

1973) and Bl. sulcata (Kaufmann 1966) into aggregations.

The defensive secretions of both species are mainly com-

posed of methyl- and ethyl-1,4-BQ and 1-tridecene

(Peschke and Eisner 1987; Tschinkel 1975a), but it is still

speculative which of them function as active agents.

Within the Stizopina, E. opacus uses the monoterpenes to

locate the burrows of P. armaticeps (Geiselhardt et al.

2006a). (-)-Camphene alone elicits as much attraction as

the complete monoterpene blend, but neither (-)-a-pinene,

(-)-b-pinene, nor (-)-limonene are active as single com-

pounds. Although it is still open whether all Stizopina use

monoterpenes as aggregation pheromone, this is most

likely, as the monoterpenes are the only specific charac-

teristic for the whole subtribe. 1,4-BQs and 1-alkenes are

almost ubiquitous in the defensive secretions of darkling

beetles (Brown et al. 1992; Tschinkel 1975a). Thus, they

are not sufficient for the discrimination against non-stiz-

opinid tenebrionids, as it was observed by Rasa (1994). In

addition, 1-alkenes are too variable in their quantitative

and qualitative composition to serve as a common signal

for the whole subtribe.

In spite of the fact that the defensive secretions function

as a general aggregation pheromone for the whole subtribe,

interspecific associations between different Stizopina spe-

cies are rare and much more limited than it would be

expected from their co-occurrence in the same habitat

(Rasa and Endrody-Younga 1997). In the Kalahari desert,

Am. mesoleius and Pl. cribricollis co-occur with E. opacus

and P. a. armaticeps, but they are never found together

with the latter, although they are attracted to the secretion

of P. a. armaticeps. Geiselhardt et al. (2006b) have shown

that guarding males of P. a. armaticeps are able to dis-

tinguish intruders to the burrow by means of their cuticular

hydrocarbons. The cleptoparasite E. opacus shows a

hydrocarbon profile that is very similar to that of its host

P. a. armaticeps, whereas the profiles of Am. mesoleius and

Pl. cribricollis differ markedly (Geiselhardt et al. 2006b,

Fig. 1 Distribution of the largest aggregations of Adoryacus bidens(Adbi), Amathobius mesoleius (Amme), Blenosia namaquensis(Blna), Eremostibes barbatus (Erba), Eremostibes bushmanicus(Erbu), Eremostibes opacus (Erop), Parastizopus a. armaticeps(Paaa), Planostibes cribricollis (Plcr), and Planostibes namaqua(Plna) between shelters with conspecific (black), heterospecific (grey)

or without (white) defensive secretion (N = 15). Asterisks indicate

significant preferences for shelters with defensive secretion

(*** P \ 0.001, v2 test)

Chemical composition and pheromonal function of the defensive secretions in the subtribe Stizopina 5

unpublished data). This may explain the acceptance of

E. opacus and the dispossession of the other two species.

Although the defensive secretion is not the crucial element

in the maintenance of interspecific associations, cross-

attraction resulting from the inability to discriminate

between different defensive secretions might represent the

initial step in the formation of interspecific associations in

the subtribe.

Acknowledgments We are grateful to Stefanie Geiselhardt and

Vanessa Zabka for their field assistance, and the Ministry of

Environment and Tourism (Republic of Namibia), the Northern Cape

Nature Conservation Service and the Western Cape Nature Conser-

vation Board (Republic of South Africa) for the granting of the

collection permits. This work was supported by the Deutsche

Forschungsgemeinschaft (Pe 231/13-1).

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