11
Morphological and Biological Evidence for the
Presence of a Male Sex Pheromone of the
Diamondback Moth
Y. S. Chow, Y. M. Lin, and H. J. Teng
Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC
Abstract
In the present study, besides morphological evidence, two bioassays, antenna-excision
and hairpencil-excision, were used to demonstrate the presence of a male sex pheromone
of the diamondback moth Plutella xylostella L. Although the antenna-excision bioassay
demonstrated that a male aphrodiasic pheromone exists in the diamondback moth, it was
proven to be not as important as the female sex pheromone in mating. After the hairpencils
of males were excised, the mating success of the females decreased significantly, suggesting
that the male hairpencils might play a role in the mating of the female diamondback moth.
introduction
Hairpencils, highly specialized scales, are present on the integument of the males
of various lepidopteran species. They are exposed during the pre-courtship rituals that
lead to various species-specific behavior patterns (Grant and Brady 1975, Baker and
Carde 1979, Rutowski 1980, Hirai et al 1981). This has been studied in tobacco budworm
(Heliothis virescens). The release of an airborne chemical by these organs suppresses
the emission of the sex pheromone secreted by the female (Hendricks and Shaver 1975).
However, Clearwater (1972) reported that Mythimna separate (Walker) produced a
pheromone from hairpencils and suggested that the major component, benzaldehyde,
acted as an arrestant that prevented the escape of the female during courtship. On the
other hand, Gothilf and Shorey (1976) found that in cabbage looper (Trichoplusia ni)
the scent of the male moth was not an essential component in courtship behavior.
Recently Baker et al (1981) and Nishida et al (1982) found that a blend of ethyl transcinnamate, methyl 2-epijasmonate, methyl jasmonate, and (R)-(-)-mellein, identified
from the hairpencils of male oriental fruit moth, attracts sex pheromone-releasing females
several centimeters away.
The objectives of the present study were to determine the effect of the hairpencils of the male diamondback moth (DBM), Plutella xylostella L (Lepidoptera:
Yponomeutidae) on mating and to demonstrate the presence of a male sex pheromone,
if any.
Materials and Methods
Experimental insects
The DBM adults were obtained either from a laboratory where they were reared
on kale seedlings, or from field population on cabbage and kale during the pupal or
late larval stages. The pupae were kept individually in a 4.5 c mx 1 cm (diam) vial
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Chow, Lin, and Teng
maintained at 25 ±1°C in a 16:8 light-dark photoperiod incubator. Newly emerged presexed adults were maintained individually in the same incubator. A 5% sugar solution
was provided as food to the adults.
Morphology
Hairpencils were exposed by squeezing the base of the male abdomen and removed
with fine forceps. They were fixed by immersion in fixative (3% glutaraldehyde), and
then transferred to amyl acetate to facilitate critical pointed drying. They were coated
with a 50 nm film of gold in a Polaron Sputter Coater and examined and photographed
using a Hitachi 450 Scanning Electron Microscope (Chow et al 1976). For light
microscopic observation, the male abdomen tip was dissected out and fixed in Zenker’s
solution for two hours, dehydrated, and embedded in paraffin. Serial sections, 6-10 u
thick, were cut and stained with Harris’s hematoxylin as described previously (Chow
et al 1976).
Bioassay
Male mating frequency per night One to three day-old adults were used. One male
and five female moths were kept in each of the 14, 17 c m x 8 cm (diam) containers.
All containers were placed overnight (about 14 h) in a dark room at 22 ± 1°C. The females
were anatomized the following day to check for the presence of sphermatophores and
the size of bursa copulatrix to judge the success of mating (Yang and Chow 1978,
Fujiyoshi et al 1979).
The antenna-excision method One half to four day-old adults were used. The insects
were anesthetized by ether. Ten males with excised antennae were paired with 10 normal
females, 10 females with excised antennae were paired with 10 normal males, and 10
pairs of adults that served as a control were kept in 17 c m x 8 cm (diam) containers
separately. All were kept in a dark room at 19 ± 3°C overnight. The presence of
spermatophores and the size of bursa copulatrix were used to judge mating success on
the following day (Yang and Chow 1978, Fujiyoshi et al 1979). The data were analyzed
by Duncan’s multiple range test.
The hairpencil-excision method One half to four day old male moths were separated
into four groups. Each group consisted of 19 pairs. In the first group, male moths were
anesthetized by ether. Then with the help of forceps covered with a polyethylene tubing
the tip of abdomen was pressed tenderly until the hairpencils came out. With another
pair of forceps the hairpencils were pulled out.
The second group of insects were treated similarly to the first except that the tip
of hairpencil tube was sealed with glue to prevent the secretion of sex pheromone from
the inside of the tube. This is termed the tube-sealing group.
The third group of insects was treated in the same way as the first except that
the hairpencils were not touched.
The fourth group of insects were normal adult males.
Each group of insects was maintained in a 17 cm x 8 cm (diam) container. A 5 %
sugar solution adsorbed on sponge was placed in each and the containers were covered
with cheesecloth to allow ventilation. All four groups were kept in a dark room at 19 ±2°C
overnight. The following day, the insects were dissected to check the progress of mating
by the size of bursa copulatrix and the presence of sphermatophores (Yang and Chow
1978, Fujiyoshi et al 1979). The data were analyzed by Duncan’s multiple range test.
Male Sex Pheromone in DBM
105
Results and Discussion
Morphology
The photomicrograph of artificially extruded hairpencils and accompanying scent
brush glands of the male DBM are shown in Figure 1 and the normal non-exposed
hairpencils in Figure 2. Morphologically speaking, these hairpencils were identical to
those of cabbage looper moth described by Gothilf and Shorey (1976) and of the flour
moth described by Corbet and Fook (1977). In Figure 2, small droplets within the
hairpencils are easily seen. It is suspected that they secrete the pheromone. Whether the
droplets observed by us are identical to small vesicles in noctuid male moths observed
by Clearwater (1975) still needs further study.
Figure 1. a. A scanning electronmicrographof the exposed hairpencils (arrow) of the male DBM
showing the position on the abdomen, and the genital clasper (C) (100x).
b. Enlarged view of the exposed hairpencils (500x).
c. and d. Enlarged view of
the exposed scent brush gland (3000x and 15000x)
Bioassay
Mating frequency A male moth mated with only one female per night. This was
confirmed by the observation of the size of bursa copulatrix and the presence of
spermatophores within bursa copulatrix in females.
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Chow, Lin, and Teng
Figure 2. a. A scanning electronmicrograph of the non- exposed hairpencils (arrow) and the
infold scent brush gland.
b. Photomicrograph of the hairpencils and their scent
c. and d. Same as b, but with greater magnification
brush gland (100x).
(200x and 400x) showing the small droplets within the hairpencils
The antenna-excision method The effect of antenna-excision on mating success is
presented in Table 1 . The average number of females with spermatophores in the control
group was significantly greater than in antenna-excision groups. The number of moths
with spermatophores in the female antenna-excision group was significantly greater than
in the male excision group. Since electroantennograms (EAG) have been used extensively
to identify the female or male sex pheromones (Grant et al 1972, Jacobson et al 1976,
Chow et al 1980), the antenna is believed to be the only olfactory receptor of sex
pheromone. It has also been established that female sex pheromone in many lepidopteran
species, including DBM, plays an important role in mating success. In the present study,
our results confirmed this. When the antennae of male moths were excised, the mating
success decreased to almost nil. When the antennae of females were excised, the mating
success decreased significantly but not as drastically as when antennae of males were
excised. This indicates that a male sex pheromone was involved in mating success but
it was not as important as the female sex pheromone.
Table 1. The effect of antenna-excision on mating of DBM
Treatment
Mean number of females/10
with spermatophores
Control
8.6a
5.8b
Female without antenna
0.4c
Male without antenna
Means followed by the same letter are not significantly different at
5% level according to Duncan's multiple range test.
The hairpencil-excision method The results of the effect of hairpencil-excision
on mating success are presented in Table 2. The average number of females with
spermatophores in the normal group was not significantly different from that in the
control group. Both groups were significantly different from the hairpencil-excision
Male Sex Pheromone in DBM
107
group and the tube-sealing group. The tube-sealing group was not significantly different
from the hairpencil-excision group.
Table 2. Effect of hairpencil-excision and tube-sealing
on mating of DBM
Mean number of females/10
with spermatophores
Treatment
Normal
Control
Without hairpencils
Tube-sealing
8.18a
6.18a
3.36b
3.09b
Means followed by the same letter are not significantly different
at 5 % level according to Duncan's multiple range test.
In this part of the study, when the hairpencils were excised, the average matings
decreased considerably compared with the normal and control groups. This showed that
the hairpencils of DBM do play a very important part in mating success. The success
of mating of some pairs suggests that either the hairpencils were not pulled out completely
or that other scent glands present in other parts of body, such as small scent glands
and wing glands which are present in male cabbage looper (Grant 1971), are involved
in mating. Further histological studies are necessary to define their involvement.
Mating success of moths in the tube-sealing group did not differ significantly from
those without hairpencils. This could be for the following two reasons. Firstly, the
quantity of male sex pheromone did not reach the behavior response threshold. Naturally
when the hairpencils are displayed before a female moth it secretes a large quantity of
male sex pheromone. But when the hairpencils were pulled out, no such action has been
involved. Therefore, there is no difference. Secondly, the glue probably cannot
completely prevent the escape of volatile sex pheromone chemicals of the male moth.
Both antenna-excision and hairpencil-excision methods proved the presence of a
male sex pheromone in DBM. Further studies involving electroantennogram and isolation
and identification of the pheromone structure are needed to reach an understanding of
the vital function of hairpencils.
Acknowledgement This study was supported by the National Science Council,
Taiwan, ROC (NSC73-0201-B001-27).
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