Journal of Chemical Ecology, Vol. 31, No. 11, November 2005 ( #2005)
DOI: 10.1007/s10886-005-7622-x
IDENTIFICATION OF A SEX PHEROMONE FROM MALE
YELLOW MEALWORM BEETLES, Tenebrio molitor
GARETH P. BRYNING,* JOHN CHAMBERS,
and MAUREEN E. WAKEFIELD
Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK
(Received March 15, 2005; accepted July 12, 2005)
Abstract—The sex pheromone released by the adult female Tenebrio molitor,
4-methyl-1-nonanol, is well known. In addition, there is evidence that adult
males release a pheromone that attracts females. The purpose of the present
study was to isolate and identify male-released pheromone(s). Emissions from
virgin adult males and females were collected on filter paper and extracted
with pentane. Extracts were analyzed by gas chromatography-mass spectrometry. One male-specific compound was detected and identified as (Z )-3dodecenyl acetate (Z3-12:Ac). In arena bioassays, E3-12:Ac was attractive to
females only, at 1 and 10 2g doses. E3-12:Ac was also attractive to females
at a 10-2g dose. The presence of both male and female pheromones, each
attracting the opposite sex, may contribute to maintaining a high-density
population of both sexes.
Key WordsV4-methyl-1-nonanol, bioassay, Coleoptera, (E)-3-dodecenyl
acetate, electroantennography, male sex pheromone, Tenebrio molitor,
(Z )-3-dodecenyl acetate.
INTRODUCTION
Tenebrio molitor L. (Coleoptera: Tenebrionidae) is a cosmopolitan secondary
pest and scavenger of many stored cereal products. T. molitor are also found in
chicken and other bird houses where feathers, food, and excrement are mixed
(Cotton, 1956). The larvae of T. molitor are widely available as pet food for
birds, fish, and reptiles (Rees, 2004).
Adult male and female mealworms both produce sex pheromone(s) that
elicit responses from the opposite sex (Happ and Wheeler, 1969; Smart et al.,
* To whom correspondence should be addressed. E-mail: [email protected]
2721
0098-0331/05/1100-2721/0 # 2005 Springer Science + Business Media, Inc.
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BRYNING ET AL.
1980). The sex pheromone released by the adult female, 4-methyl-1-nonanol, is
well known (Tanaka et al., 1986).
Adult males release a pheromone that attracts females (Valentine, 1931;
Tschinkel et al., 1967; Happ, 1969; August, 1971; Tanaka et al., 1986), and
possibly also an antiaphrodisiac that inhibits the response of other males to the
female scent (Happ, 1969). August (1971) reported that locomotion of females
decreased in close proximity to males in response to a male-produced pheromone,
resulting in females aggregating near live males. If the live male was moved to
another chamber of the olfactometer, females reaggregated in this chamber within
an hour. Live males or extracts thereof did not cause aggregation of males. The
purpose of the present study was to identify the volatile pheromone(s) produced
by male T. molitor that elicited this aggregation behavior.
METHODS AND MATERIALS
Insects. Tenebrio molitor, a laboratory strain kept in culture since 1958,
was cultured on a 5:5:1 mixture by weight of wheatfeed, rolled oats, and yeast
at 25-C, 70% RH, with a 16L:8D photoperiod. Insects were sexed at the pupal
stage (Bhattacharya et al., 1970), kept separately, and tested as adults between
8 and 25 d after pupal eclosion.
Chemicals. 4-Methyl-1-nonanol was synthesized by LiAlH4 reduction of
4-methylnonanoic acid (purity 99%; Aldrich Flavors and Fragrances, UK).
(Z)-3-Dodecenyl acetate (Z3-12:Ac; isomeric purity 99%) and (E )-3-dodecenyl
acetate (E3-12:Ac; isomeric purity 99%) were obtained from Pherobank, Research
Institute for Plant Protection, The Netherlands. Pentane (glass distilled grade)
was from Rathburns (UK). Cylclohexane (purity 99.99%) was from Aldrich (UK).
Preparation and Analysis of Extracts. Two sexed groups of five to six
virgin male and female adults (8Y25 d after pupal eclosion) were kept in glass
jars (50 50 mm) for 24 hr at 20-C in a 16L:8D photoperiod with one 50-mmdiam disk of filter paper (Whatman qualitative No.1) placed in the lid of each
jar. The filter papers were then placed into separate 5-ml Supelco glass microreaction vessels and extracted with 3 ml of pentane for 30 min, and the
extracts concentrated to 50 2l by using a stream of nitrogen. A group of virgin
males (N = 11) was also directly extracted with 5 ml of pentane for 1 hr and the
extract was concentrated to 100 2l with a stream of nitrogen. This extract was
used for ozonolysis of the male extract, and determination the stereochemistry
of the male pheromone.
Extracts were analyzed on a Hewlett-Packard 5890 series II coupled to a VG
Trio-1 mass spectrometer (MS). Aliquots of the extracts (3 2l) were injected in
splitless mode ( purge on after 1 min) at 250-C using helium as a carrier gas in
IDENTIFICATION OF A SEX PHEROMONE FROM MALE YELLOW MEALWORM BEETLES
2723
constant flow mode. A 50-m Chrompack CP-Sil 19CB column (0.25 mm i.d., film
thickness 0.2 2m) was used except for the determination of the stereochemistry
of the pheromone, where a CP-Wax 52CB column was used (0.25 mm i.d., film
thickness 0.2 2m). The GC was programmed from 50-C 1 min to 270-C at 10-C/
min, and maintained for 20 min. The MS was operated in electron impact
ionization mode (EI, 70 eV) and scanned from 33 to 650 amu once every second.
The MS and interface temperatures were 200 and 275-C, respectively.
Ozone was bubbled into a 5-ml glass microreaction vessel containing an
extract of males in pentane for 1 min. An aliquot (3 2l) of the resulting extract was
then analyzed by GC-MS on a CP-Sil 19CB column. The extract was repeatedly
ozonolyzed until the male produced GC peak completely disappeared.
Authentic standards of Z3-12:Ac and E3-12:Ac were analyzed by GC-MS
using the CP-Wax 52CB column. The oven was programmed from 50-C 1 min
to 120-C at 8-C/min, hold for 27 min, then to 250-C at 10-C/min. An aliquot of
an extract of males was analyzed under the same conditions.
Electroantennogram Studies. Prior to testing Z3-12:Ac in behavioral
bioassays, the electroantennogram (EAG) response of both male and female
T. molitor to Z3-12:Ac and E3-12:Ac was examined. Whole T. molitor proved
difficult to restrain so head preparations were used, similar to EAG studies with
other coleopteran species (e.g., Okada et al., 1992; Scholz et al., 1998).
Five male and five female adult T. molitor (less than 3 wk post-eclosion)
were tested. Insects were removed from a culture approximately 1 hr prior to
testing. The head was removed and secured using double-sided adhesive tape.
The recording and indifferent electrodes were positioned in the terminal
antennal segment and in the neck cavity, respectively. Electrodes were filled
with saline (Roelofs, 1984) and connected, using chloridized silver wires, to a
Grass P16 preamplifier and a chart recorder.
The odor delivery system was as described by White and Birch (1987). Z312:Ac and E3-12:Ac were presented at five doses ranging from 1 ng to 10 2g in
10 2l of cyclohexane. The test materials were presented at ca. 2-min intervals in
ascending concentration and at each concentration the order of presentation was
random. To account for variations due to fatigue, a standard of carob extract
prepared from Porapak-Q collected volatiles from kibbled carob pods, was used
(Wakefield et al., 2005). The standard was tested at the start and the end of each
test and after presentation of each log dose of the two isomers. The absolute net
EAG response to a test material or the standard was determined by first subtracting the mean response to a solvent control stimulation at the start and the
end of each test from the response to the sample. Results were then expressed as
a percentage of the response to the standard by:
Relative net EAGx ¼
2 absolute net EAGx
100
absolute net standardx þ standardxþ1
2724
BRYNING ET AL.
Responses of males and females were compared using Student’s t test. The
perception threshold, defined as the lowest amount at which the absolute EAG
response to the test stimulus was significantly greater than the mean of the
control responses, was also determined.
Behavioral Bioassays. Pupae were removed from the culture and kept at
22-C and 16L:8D photoperiod, in individual glass tubes (75 25 mm) with a
perforated plastic lid for ventilation and a folded filter paper as a crawling
surface. The bioassay was adapted from the method described by Finnegan
and Chambers (1993). Individual insects were placed in an arena comprised a
20-cm-diam metal ring on filter paper circles (27 cm diam, Whatman No. 1) and
covered with a glass plate. The rings were 40 mm high and narrowed toward the
top, making a considerable overhang for the insects to climb, thus preventing
escape. Insects were allowed to settle for 30 min. One insect was introduced
into the center of each arena. With the beetle at the perimeter of the arena, one
20-mm-diam filter paper disk (Whatman No.1) treated with test material in
10 2l of pentane and a control disk treated with 10 2l of pentane were placed
25 mm apart, equidistant from the center of the arena along a diameter perpendicular to the insect. The solvent was allowed to evaporate from the disks
for 30 sec before they were placed in the arenas. The disk which was touched
first was recorded (= a behavioral response). Bioassays were run for 10 min.
Except where stated otherwise, each dose of each test material was tested
against 40 adults of each sex.
Doses of 100 ng, 1 2g, or 10 2g of Z3-12:Ac in 10 2l of pentane were
applied to test filter papers and tested against both males and females. E3-12:Ac
was tested against females only at (10 2g), so that its biological activity could
be compared with that of Z3:12:Ac, even though E3-12:Ac was not found in
male extracts. The responses of males and females to each dose of Z3-12:Ac,
and E3-12:Ac versus the controls were analyzed using # 2 test with Yates’
correction for 1 degree of freedom.
RESULTS
Identification of Material Produced by Males. The extract of filter papers
exposed to females contained 4-methyl-1-nonanol, previously identified as a
sex pheromone released by females (Tanaka et al., 1986). Its identity was confirmed by matching its mass spectrum and retention time on the CP-Sil 19CB
column with those of an authentic standard.
Extracts of filter papers exposed to males (Figure 1C) contained a
compound (peak 2) that was not detected in extracts of blank filter papers
(Figure 1A), or filter papers exposed to females (Figure 1B). This compound
gave a fragmentation pattern (Figure 2A) with ions at m/z 61, 96, 109, 124, 138,
IDENTIFICATION OF A SEX PHEROMONE FROM MALE YELLOW MEALWORM BEETLES
2725
FIG. 1. Total ion chromatograms of extracts of filter papers which were (A) blank,
(B ) exposed to five females, and (C) exposed to five males. Identifications of peaks:
(1) 4-methyl-1-nonanol, (2) Z3-12:Ac. CP-Sil 19CB column.
FIG. 2. (A) EI and (B ) CI mass spectra of male produced compound.
2726
BRYNING ET AL.
TABLE 1. MEAN EAG RESPONSES (T SE) IN MICROVOLTS TO Z3-12:AC AS A
PERCENTAGE OF THE RESPONSE TO A CAROB EXTRACT STANDARD (N = 5)
Dose
1 ng
10 ng
100 ng
1 2g
10 2g
Female
j4.5
3.0
23.6
7.8
40.5
T
T
T
T
T
2.1
3.4
8.4
8.7
4.1*
Male
0.5
0.8
6.2
35.4
21.9
T
T
T
T
T
3.7
3.9
6.0
13.8
4.0*
*Significant difference (t = 3.28, df = 8, P < 0.05) between the means for males and females.
and 166, consistent with a dodecenyl acetate. The CI (isobutane) spectrum
showed an ion at m/z 227 (M + H)+, confirming the molecular weight as 226
(Figure 2B). After ozonolysis of the extract, peak 2 disappeared, confirming that
the compound was unsaturated. A new peak appeared at scan 299 and was identified as nonanal from diagnostic fragment ions at m/z 142 (M +, 0.8), 124 (3),
114 (8), 98 (35), 82, (31), 70 (41), 57 (85), 55 (72), 43 (79), and 41 (100%).
This indicated that the double bond was in the 3-position in the starting
material. Authentic Z3-12:Ac and E3-12:Ac eluted at scans 2071 and 2056
respectively, on the CPWAX-52CB column. The compound of interest in the
extract from males eluted at scan 2070, confirming the material as Z3-12:Ac.
No E3-12:Ac was detected in the male extract.
Electroantennography. The mean responses to the carob standard based on
the first presentation of the standard were 305 T 45.5 and 183.5 T 40.8 2V for
females and males, respectively (N = 5). Based on the final presentation of
the carob standard, the mean responses to the standard were 430 T 79.6 and
257.5 T 71.9 2V for females and males, respectively (N = 5). There was no
significant difference (ANOVA, F = 2.81, P > 0.05, df = 19) between the first
response and the final response to the carob standard or for the response by
males and females at either point.
TABLE 2. MEAN EAG RESPONSES (T SE) IN MICROVOLTS TO E3-12:AC AS A
PERCENTAGE OF THE RESPONSE TO A CAROB EXTRACT STANDARD (N = 5)
Dose
1 ng
10 ng
100 ng
1 2g
10 2g
Female
j3.1
2.1
2.0
12.8
22.6
T
T
T
T
T
2.5
3.5
5.8
8.8
8.0
Male
0.1
1.6
32.3
1.5
39.2
T
T
T
T
T
2.2
4.3
15.5
4.3
22.3
2727
IDENTIFICATION OF A SEX PHEROMONE FROM MALE YELLOW MEALWORM BEETLES
TABLE 3. TWO-CHOICE BIOASSAY RESPONSES OF MALE
Z3-12:AC (N = 40)
AND
FEMALE T. molitor
TO
Percent responsea
Dose
Males
100 ng
1 2g
10 2g
Females
100 ng
1 2g
10 2g
Z3-12:Ac
Control
No response
37.5
55
55
25
40
35
37.5
5
10
50
65*
75**
35
20
20
15
15
5
# test: #2 = 9.53, df = 1, P = 0.002 (*); #2 = 12.74, df = 1, P < 0.001 (**).
a 2
The mean EAG responses of female and male T. molitor to Z3-12:Ac and
E3-12:Ac as a percentage of the response to the standard are shown in Tables 1
and 2, respectively. There was no difference in response by males and females
except for 10 2g of Z3-12:Ac, where the response by females was greater than
the response by males (t = 3.28, P < 0.05, df = 8). The perception threshold for
both Z3-12:Ac and E3-12:Ac by females was 10 2g (P < 0.05, df = 4). In males,
the response to Z3-12:Ac and E3-12:Ac did not differ significantly from the
control response at the quantities tested (P > 0.05, df = 4).
Behavioral Bioassay Tests. Z3-12:Ac was not attractive to males at any
dose tested (# 2 test, P > 0.05) (Table 3). However, at 1 and 10 2g, Z3-12:Ac was
attractive to females (P = 0.002 and P < 0.001, respectively) (Table 3). E3-12:
Ac was also attractive to females at 10 2g (62.5% of females responded to E312:Ac and 22.5% to the control; # 2 = 7.53, df = 1, P = 0.006).
DISCUSSION
Males of many beetle species that infest stored products produce aggregation pheromones (Chambers, 1991). In contrast, Z3-12:Ac is a male-produced
sex pheromone that elicits a response only from females. August (1971) tested
live males and extracts of males over a range of doses in a choice test and found
that one male equivalent of male extract caused attraction of females into a
central chamber and the response was dose-dependent. In our study, in the
highest dose of Z3-12:Ac tested (10 2g), approximately five male equivalents
was highly attractive to females. August (1971) found that male extracts were
not attractive to males. Similarly, we found that Z3-12:Ac was not attractive to
males at any dose tested.
2728
BRYNING ET AL.
August (1971) also found that the response of females to live males (where
the males were not visible to the females) was greater than to the optimally
attractive dose of male extract (5 male equivalents). He suggested that this
could be due to other semiochemicals present in the male extracts, which might
be repellent to females at high concentrations. Happ and Wheeler (1969) found
that males release an antiaphrodisiac after they are exposed to female scent,
which reduces the attractancy of the female scent to subsequent males. When
they tested a mixture of male and female scent against males, it was considerably much less attractive than the female scent alone. They also found that
females often extruded their ovipositors on exposure to male sex attractant, and
suggested that, together with the antiaphrodisiac component, males increase the
probability that females will utilize their sperm before another male has a
chance to mate. The male semiochemical(s) suggested by August (1971) and the
antiaphrodisiac reported by Happ and Wheeler (1969) could be the same
compound(s). Here, we found evidence for the production by males of 2,5dimethylpyrazine, but the amounts were very small and we did not determine
whether this compound has any biological activity in T. molitor.
E3-12:Ac (98%) and Z3-12:Ac (2%) are the two components of the sex
pheromone of the potato tuber moth, Scrobiphalpopsis solanivora Povolny
(Nesbitt et al., 1985). E3-12:Ac has also been identified as the sex pheromone of
Scrobipalpa ocellatella Boyd (Renou et al., 1980). These are the only other
species of insects reported to use E3-12:Ac or Z3-12:Ac as semiochemicals.
Many species of moths are reported to use one or more isomers of dodecenyl
acetate as components of their female sex pheromones, but to our knowledge,
this is the first report of a dodecenyl acetate as a beetle pheromone.
It is unusual for both sexes of a species to release a pheromone that attracts
the other sex. Both sexes of the Azuki bean weevil (Callosobruchus chinensis
L.) release a nonvolatile, copulatory release pheromone, which affects only
males and is distinct from the female-released sex pheromone (Tanaka et al.,
1981). Male and female Tribolium confusum (the confused flour beetle) release
three hydrocarbons which increase the frequency of copulation, but which affect
only males (Keville and Kannowski, 1975). In contrast, the pheromone identified in our study acts as a sex attractant for females, as opposed to a contact
copulatory release pheromone.
Studies of T. molitor mating behavior by Obata and Hidaka (1982) found a
low rate of successful copulation due to relatively random mounting behavior
by males, and suggested that a Bcritical^ high-density population is needed to
overcome this. A high-density Bcrowded state^ was reported to accelerate
oocyte development, pheromone emission, and mating success (Mordue, 1965;
Happ and Wheeler, 1969; Gerber, 1973). As females do not respond to their
own pheromone, a female pheromone would only lead to an increase in the
density of males, and not of both sexes. The presence of both male and female
IDENTIFICATION OF A SEX PHEROMONE FROM MALE YELLOW MEALWORM BEETLES
2729
pheromones, each attracting the opposite sex may, therefore, contribute to
maintaining a high-density population of both sexes.
AcknowledgmentsVWe thank Dr. Larissa Collins and Mr. A. Prickett for statistical analysis of
the behavioral bioassay results. This work was funded under the CSL Seedcorn budget by the UK
Department for the Environment, Food, and Rural Affairs.
REFERENCES
AUGUST, C. J. 1971. The role of male and female behaviour of Tenebrio molitor. J. Insect Physiol.
17:739Y751.
BHATTACHARYA, A. K., AMEEL, J. J., and WALDBAUER, G. P. 1970. A method for sexing living
pupal and adult yellow mealworms. Ann. Entomol. Soc Am. 63:1783Y751, 1970.
CHAMBERS, J. 1991. Overview of stored-product insect pheromones and food attractants. J. Kans.
Entomol. Soc. 63:490Y499.
COTTON, R. T. 1956. Pests of Stored Grain and Grain Products. Burgess Publishing, Minneapolis,
MN.
FINNEGAN, D. and CHAMBERS, J. 1993. Identification of the sex pheromone of the Guernsey carpet
beetle, Anthrenus sarnicus Mroczkowski (Coleoptera: Dermestidae). J. Chem. Ecol. 19:
971Y983.
GERBER, G. H. 1973. Reproductive behaviour and physiology of Tenebrio molitor (Coleoptera:
Tenebrionidae). I. Initiation of mating in young adults and the effects on adult density. Can.
Entomol. 105:807Y811.
HAPP, G. M. 1969. Multiple sex pheromones of the mealworm beetle, Tenebrio molitor L. Nature
222:180Y181.
HAPP, G. M. and WHEELER, J. 1969. Bioassay, preliminary purification, and effect of age, crowding,
and mating on the release of sex pheromone by female Tenebrio molitor. Ann. Entomol. Soc.
Am. 62:846Y851.
KEVILLE, R. and KANNOWSKI, P. B. 1975. Sexual excitation by pheromones of the confused flour
beetle. J. Insect Physiol. 21:81Y84.
MORDUE, W. 1965. Studies on oöcyte production and associated histological changes in the
neuroendocrine system in Tenebrio molitor L. J. Insect Physiol. 11:493Y503.
NESBITT, B. F., BEEVOR, P. S., CORK, A. A., HALL, D. R., MURILLO, R. M., and LEAL, H. R. 1985.
Identification of components of the female sex pheromone of the potato tuber moth,
Scrobipalpopsis solanivora. Entomol. Exp. Appl. 38:81Y85.
OBATA, S. and HIDAKA, T. 1982. Experimental analysis of mating behavior in Tenebrio molitor L.
(Coleoptera: Tenebrionidae). Appl. Entomol. Zool. 17:60Y66.
OKADA, K., WATANABE, A., MORI, M., SCHIMAZAKI, K., CHUMAN, T., MOCHIZUKI, F., and
SHIBUYA, T. 1992. Olfactory responses to the sex pheromone component and its behavioural
inhibitor in the male cigarette beetle, Lasioderma serricorne. J. Insect Physiol. 38:705Y709.
REES, D. 2004. Insects of Stored Products, pp. 112Y113. Manson Publishing, London.
RENOU, M., DESCOINS, C., LALLEMAND, J. Y., PRIESENER, E., LETTERE, M., and GALLOIS, M. 1980.
L’acetoxy-1-dodecene 3E, composant principal de la pheromone sexuelle de la teigne de la
betteravae: Scrobipalpa ocellatella Boyd. Z. Angew. Entomol. 90:275Y289.
ROELOFS, W. L. 1984. Electroantennogram assays: Rapid and convenient screening procedures for
pheromones, pp. 131Y160, in H. E. Hummel and T. A. Miller (eds.). Techniques in Pheromone
Research. Springer-Verlag, New York.
2730
BRYNING ET AL.
SCHOLZ, D., BORGEMEISTER, C., and POEHLING, H. M. 1998. EAG and behavioural responses of the
larger grain borer, Prostephanus truncatus, and its predator, Teretriosoma nigrescens, to the
borer-produced aggregation pheromone. Physiol. Entomol. 23:265Y273.
SMART, L. E., MARTIN, A. P., and CLOUDSLEY-THOMPSON, J. L. 1980. The response to pheromones
of adult and newly emerged mealworm beetles (Tenebrio molitor L.) (Coleoptera: Tenebrionidae). Entomol. Mon. Mag. 116:139Y145.
TANAKA, K., OHSAWA, K., HONDA, H., and YAMAMOTO, I. 1981. Copulation release pheromone,
Erectin, from the Azuki bean weevil (Callosobruchus chinensis L.). J. Pest. Sci. 6:75Y82.
TANAKA, Y., HONDA, H., OHSAWA, K., and YAMAMOTO, I. 1986. A sex attractant of the yellow
mealworm, Tenebrio molitor, and its role in mating behaviour. J. Pest. Sci. 11:49Y55.
TSCHINKEL, W., WILSON, C., and BERN, H. A. 1967. Sex pheromone of the mealworm beetle
(Tenebrio molitor). Exp. Zool. 164:81Y85.
VALENTINE, J. M. 1931. The olfactory sense of the adult mealworm beetle, Tenebrio molitor ( Linn.).
J. Exp. Zool. 58:165Y227.
WAKEFIELD, M. E., BRYNING, G. P., COLLINS, L. E., and CHAMBERS, J. 2005. Identification of
attractive components of carob volatiles for the foreign grain beetle, Ahasverus advena (Watl )
(Coleoptera: Cucujidae). J. Stored Prod. Res. 41:239Y253.
WHITE, P. R. and BIRCH, M. C. 1987. Female sex pheromone of the common furniture beetle
Anobium punctatum (Coleoptera: Anobiidae): extraction, identification and bioassays. J. Chem.
Ecol. 13:1695Y1706.