SEX PHEROMONES OF THE SCARABAEIDAE
Meg Donohue
April 1994
EN 570
Colorado State University
Fort Collins, Colorado 80523
Abstract
Sex Pheromones of the Scarabaeidae
The Scarabaeidae is a large family of beetles (Coleoptera)
containing nearly 1400 North American species alone. The members vary
greatly in size, color, and behavior. They are normally heavily
scleroterized and convex in shape (Borror et. al. 1989). Scarabs, both
in the adult and larval stage, are a costly economic pest due to the
damage they are able to inflict above ground on plants, crops, and trees
and below ground on roots (Potter and Haynes 1993, Tumlinson et. al.
1977).
As concern for preserving the environment increases integrated
pest management (IPM) approaches are replacing insecticides in
controlling insect populations. One IPM approach is the use of sex
pheromones. Chemical cues emitted by one sex of a species to attract the
other are used as a means of luring the pest before it does considerable
plant damage (Leal et. al. 1992, Potter and Haynes 1993). To date there
have been few sex pheromone identifications made in the Scarabs (Mayer
and Mclaughlin 1991); however, as new approaches are explored, knowledge
of their sex pheromones will become increasingly important and valuable.
Sex pheromones or components of sex pheromones have been
identified in the Japanese beetle (Popillia japonica Newman), grass grub
beetle (Costelytra zealandica White), soybean beetle (Anomala rufocuprea
Motschulsky)(Mayer and Mclaughlin 1991), large black chafer (Holotrichia
parallela Motschulsky) (Leal et. al. 1993), Oriental beetle (Blitopertha
orientalis) (Leal 1993), cupreous chafer beetle (Anomala cuprea Hope)
(Leal 1991), Anomala schonfeldti Ohaus (Leal 1992), and Anomala daimiana
Harold (Leal 1993) (Table 1).
Introduction
Scarab sex pheromone identification is fast becoming a necessary
component of integrated pest management practices as concern for the
environment increases. Identification and isolation of these compounds
as well as accurate evaluations of bioassays are becoming an integral
part of understanding sex pheromone mechanisms and how they can be
manipulated into IPM programs. It has been recently discovered that
some Scarab sex pheromones contain more than one compound (Leal 1993,
Leal et. al. 1993). This paper will describe some of the chemical
approaches used in identifying Scarab sex pheromones and the biological
assays that accompany them. It is divided into single component sex
pheromone systems and double component sex pheromone systems.
SINGLE COMPONENT SEX PHEROMONE SYSTEMS:
Sex Pheromone:
Japonilure-
(R,Z)-5-(1-decenyl)dihydro-2(3H)-furanone or
(R,Z)-5-(-)-(dec-1-enyl)oxacyclopentan-2-one
The Japanese beetle, P. japonica, sex pheromone (diagrammed below)
was identified, isolated, and synthesized in 1977 (Tumlinson et. al.
1977).
(Doolitle et. al. 1980)
The pure and synthetic (R.Z) form of the pheromone proved to be
very attractive to males however its enantiomer (S,R) and the racemic
(equal amounts of the D and L forms of the same compound) Z isomer
inhibited the male response to the female (Doolittle et al. 1980,
Tumlinson et. al. 1977). Since the identification of this pheromone a
variety of syntheses techniques have been successfully accomplished
(Koseki et al. 1993, Ebata et. al. 1992, Baskaran et.al. 1990, Doolittle
et al. 1980). The active component of the pheromone was obtained by
washing virgin female Scarabs with a non-polar solvent, benzene, to form
a workable extract. Through "mass, infrared, and nuclear magnetic
resonance spectroscopy and chemical transformations" the pheromone was
identified (Tumlinson et. al. 1977, Mayer and Mclaughlin 1991). Purity
of the benzene extracts was achieved through liquid chromatography
separation on various column types. "Semihydrogenation" of the pure
compound yielded a racemic mixture of the Z and E isomers. When
analyzed against the natural pheromone, the synthetic mixture produced
identical results (Doolittle et. al. 1980). Through bioassays it was
realized that the racemic mixture inhibited males from responding to
virgin females. When tested separately, the Z and E isomers were
effective inhibitors of the male response at varying rates (Doolittle
et. al. 1980, Tumlinson et. al. 1977). Tumlinson et. al. (1977) tested
various proportions of the enantiomers (R,Z) and (S,Z) of the sex
pheromone. In field tests males were not attracted to the pheromone
traps when less than or equal to 1% of the enantiomer mixture contained
the (S,Z) form (Tumlinson et. al. 1977). This attraction of a Scarab by
a pheromone and its inhibited response to its enantiomer is unique, thus
far being reported only in the Japanese beetle and cupreous chafer
beetle (Anomala cuprea) (Leal et. al. 1993).
Sex Pheromone:
2-(E)-nonenol
(Leal et. al. 1993)
Anomala schonfeldti Ohaus is considered one of the most damaging
turfgrass pests in Japan. Identification of its sex pheromone is hoped
to lead to its implementation in Japan's IPM projects (Hasegawa et. al.
1993). GC-elctroantennographic detector (GC-EAD) along with behavior
bioassay provided the evidence that a sex pheromone was present in this
species. Due to the small antennae size of A. schonfeldti, the bioassay
was a necessary component. When used alone, GC-EAD, was not completely
reliable (Leal et. al. 1992). Instead a combination of high resolution
gas chromatography and behavior bioassay allowed for the identification
of the sex pheromone, 2-(E)-nonenol (Hasegawa et. al. 1993). Active
extracts of the pheromone were isolated from females feeding on cherry
leaves. After extraction and separation through GC, each fraction of
the extract was eluted off the column, collected, and tested for a
response from the male A. schonfeldti. Leal et. al. (1992) were able to
locate the active zone on the chromatogram by observing the males
immediate response to the volatile. Females who had not eaten the
cherry leaves were tested and exhibited the same response. Using a
flame ionization detector (FID) a broad peak was detected in the
previously determined active area, however use of a non-polar column
produced a small peak. By testing the volatiles on different columns,
supplemented by wind tunnel bioassays, the compound was determined to
be 2-(E)-nonenol (Leal et. al. 1992).
Evaluation of this pheromone in the field revealed no significant
difference in attractiveness as measured by the numbers of males caught
in traps (placed 90cm above ground) containing different doses of the
pheromone. However, more males were caught in traps at 30cm above
ground compared to 90cm above ground (See diagram below).
Effects of the height of the traps (Sankei, JT) on the
catches of A. schonfeldti males. (Hasegawa et. al. 1993)
A suggested reason for this is difference is mating behavior. A.
schonfeldti males emerge from the soil just after sunset and fly or
crawl at low levels to locate a female to mate with (Hasegawa et. al.
1993).
Sex Pheromone:
Phenol
Over 20 years ago phenol was identified as an active component of
the grass grub beetle, Costelytra zelandica White (Henzell and Lowe
1970). Since then no other pheromone components have yet been
discovered. The active extract of the pheromone was isolated by washing
1500 female abdomens with the non-polar solvent diethyl ether. Results,
using gas liquid chromatography (GLC), mass spectroscopy (MS), and paper
chromatography were similar and indicated phenol as the active compound
in the extracts. Measurements of mass to charge ratios confirmed this
result. A simple bioassay showed that males responded to "dummy"
females treated with phenol about 80% of the time. Similarily,
pheromone traps with a phenol-water mixture captured about 222 males
while traps containing only water captured none (Henzell and Lowe 1970,
Mayer and Mclaughlin 1991).
Sex Pheromone:
Methyl (Z)-5-tetradecenoate
Only one pheromone, methyl (Z)-5-tetradecenoate, has been
identified for the soybean beetle, A. rufocuprea Motschulsky (Mayer and
Mclaughlin 1991). Isolated volatiles from field collected beetles were
used in the identification of the pheromone. Through the use of gas-
liquid chromatography, high pressure liquid chromatography (HPLC), and
mass spectroscopy, the active peak was identified and isolated as
methyl-(Z)-5-tetradecenoate. A simple bioassay proved the pheromones
attractiveness when males, present with the pheromone, elicited a
response (Mayer and Mclaughlin 1991).
Sex Pheromone:
(Z) and (E)-tetradec-7-en-2-one
Last year a sex pheromone was isolated for Blitopertha orientalis,
(Oriental beetle), a turfgrass pest of Japan, which "utilizes an even
numbered carbon chain, unbranched, and unsaturated methyl ketone" (Leal
1993) (Diagrammed below).
(Leal 1993)
Active volatiles were collected from females washed with hexane.
In lab tests males appeared attracted to this crude extract. GC (silica
gel column)- behavior bioassay used in combination, simlutaneously,
aided in the identification of the active peak as in A. schonfeldti
Through the use of different GC column types and the fact that the
pheromone did not undergo hydrolysis in the presence of potassium
hydroxide and methyl hydroxide, Leal (1993) was able to narrow the
pheromone choice to tertradec-?-en-2-one. MS and GC analysis showed two
peaks, indicating a mixture of cis and trans isomers were present which
led to the identification as (Z) and (E) tetradec-7-en-2-one.
Confirmation of the chemical mixture as a pheromone was shown in a
bioassay (Leal 1993).
Field tests, performed at the end of the season, gave preliminary
confirmation of the pheromones attractiveness to male beetles (Leal
1993).
DOUBLE COMPONENT SEX PHEROMONE SYSTEMS:
It was once believed that insect species used one compound for
attracting their mate. It was soon discovered, as in the case of
Lepidoptera and Scarabaeidae, that more than one component is present in
the pheromone (Leal 1993, Leal et. al. 1993).
Anomala cuprea (Cupreous Chafer Beetle)
A major component, (R,Z)-5-(-)-(oct-1-enyl)oxacyclopentan-2-one
and minor component, (R,Z)-5-(-)-(dec-enyl)oxacyclopentan-2-one are
considered to be the attractants in the sex pheromone system of the
cupreous chafer beetle (Diagrammed below). The minor component is the
sex pheromone of the Japanese beetle, P. japonica (Leal 1991, Leal et.
al. 1993).
Minor component Major component
(Leal et. al. 1993)
The major active component of the pheromone was extracted from
females with the non-polar solvent hexane. Using both GC and
electroantennographic detector (EAD) one active peak was observed.
Interestingly, this was the first time an EAD was used successfully with
lamellate antennae (Leal et. al. 1992). An electron impact mass
spectrum (EI-MS) was used to help determine a precise chemical make up
(Leal 1991). A GC analysis of a synthetic enantiomeric pheromone
mixture registered a 70% (R,Z) configuration and a 30% (S,Z)
configuration (Leal et. al. 1993). However, the (R,Z) configuration,
occurring in the natural pheromone, produced one peak confirming it as
the proper configuration of the true pheromone. The (S,Z) configuration
proved to be inhibitory to males (Leal et. al. 1993) similar to the
enantiomeric inhibition in P. japonica (Tumlinson 1977). Once the major
component was produced synthetically, two bioassays were performed.
First, filter paper containing the pheromone was placed in a box with
male beetles. Approximately 80% of the males elicited sexual behavior.
When using a wind tunnel, similar results occurred (Leal 1991). It
wasn't until two years later that a minor component was reported (Leal
et. al. 1993). In procedures similar to the identification of the major
sex pheromone, the minor component, japonilure, was identified.
In one A. cuprea sex pheromone release study, interestingly it was
found that one day after mating, the amount of major pheromone
significantly decreased and then over time it increased. A possible
suggestion for the change in pheromone levels may be a selective
advantage for increasing the mating probability of unmated females (Leal
et. al. 1993). Since the levels of the two pheromones change with
mating, an optimal ratio for male attractivness was not able to be
determined. However, field tests did show that having the combination
of major/minor pheromone attracted more males than the major component
alone (Leal et. al. 1993).
Because it was found that P. japonica males are inhibited by the S
enantiomer of the female sex pheromone (Tumlinson 1977) and A. cuprea
has shown the same effect, Leal et. al. (1993), recently studied the
pheromone reception mechanism in A. cuprea to see if it discriminated
between enantiomers. Two areas of olfactory sensilla were observed on
the antennae, one with pits (left side on antennae) and one with out
pits (right side of the antennae).
A. B. C.
A - shows the left (pitted) and right (non pitted) sides of the
olfactory sensilla
B - shows the pitted pores of the antenna
C - shows the the smoother non pitted area of the antenna,
pheromone sensitive area
(Leal et. al. 1993)
With the aid of scanning electron micrographs (SEM) and
transmission electron micrographs (TEM), an electrode was placed in
different areas of the antennae. After trial and error, the area
without pits (plate like) was confirmed as pheromone sensitive (Leal et.
al. 1993). Through EAD, the appearance of two spikes during testing
suggested one for the major pheromone component and one for the minor
pheromone component. TEM showed two dendtrites below the sensitive area
which added evidence for the two part sex pheromone system. Although
both the sex pheromone and its enantiomer showed a response on the EAD,
Leal et. al. (1993), hypothesized that behavioral inhibition may result
from differences in processing information.
Anomala daiminana
Anomala daiminana sex pheromone is composed of both the sex
pheromones from A. schonfeldti , 2-(E)-nonenol (1), and A.cuprea (R,Z)-
5-(-)-(oct-1-enyl)oxacyclopentan-2-one (2), (Leal et. al. 1993)
(Diagrammed below).
(Leal et. al. 1993)
The active components of the pheromone were collected from virgin
female who fed on chestnut leaves. Using GC, MS, and EAD the active
peaks were consistant with those of 2-(E)-nonenol and (R,Z)-5-(-)-(oct-
1-enyl)oxacyclopentan-2-one. Lab bioassays (using wind tunnels) and
field tests (using pheromone traps) confirmed the sex pheromone mixture
(Leal et. al. 1993).
Holotrichia parallela
The large black chafer is another Scarab species that utilizes a
two part pheromone system consisting of L-isoleucine methyl ester (LIME)
(major component) and R-(-)-linalool (minor component) (Leal et. al.
1992, Leal et. al 1993) (Diagrammed below).
Minor component
(Leal et. al 1993)
Adult beetles emerge from the soil at night. Females release
their pheromone by extruding their abdominal gland for no more than 15
minutes. The active extracts of the major component were isolated with
the solvents ether and dichloromethane. Ether was eventually chosen
because its mixture with the volatiles elicited a stronger response to
males (Leal et. al. 1993). Using GC, EAD, and MS were used to identify
the major compound. Due to lack of information, an amino acid
derivative reaction (methyl esterfication) was performed (Leal et. al.
1993). Further GC analysis led to the identification of L-isoleucine
methyl ester as the major pheromone (Leal et. al. 1993). A year later,
through similar techniques, the minor component was identified.
Although not attractive alone, (R)-(-)-linalool enhances the
attractiveness of the major component. Similar to A. cuprea, pheromone
levels fluctuated at various times during the mating season. On the
first and third night virgin females emerged, the major pheromone level
was higher compared to the second and fourth night. Laboratory
examination of beetles collected over a one week period showed that the
major pheromone decreased over time while the minor component stayed
constant. It appeared that major pheromone production "oscillated" on a
regular schedule (i.e. a "circabidian rhythm"). In field studies, the
number of males caught in pheromone traps showed peaks every other day
which correalted with the cicabidian rhythm hypothesis (Leal et. al.
1993).
Conclusions:
Popillia japonica and Anomala cuprea are the only two Scarabs thus
far to show male inhibition to their natural pheromones' enantiomer
(Tumlinson et.al. 1977, Leal et. al. 1993). The pheromones' isomer may
play an unidentified role in insect communication not yet known
(Tumlinson et. al. 1977). Study of mechanisms of insect reception (Leal
et. al. 1993) in combination with chemical anaylsis (of the enatiomers)
and behavioral bioassays are needed to answer future questions
involving chemical communication.
No minor components to the major pheromone have been discovered in
P. japonica, A. schonfeldti, C. zealandica, A. rufocuprea, and B.
orientalis. A suggestion may be that satisfactory field results (male
Scarab beetle attraction to the natural or synthetic pheromone) often
cover-up the presence of minor pheromone components (Leal et.al. 1993).
A point of interest may be why some species in the same genus have
single component pheromone systems while others have multiple component
systems. Is this possibly due to no further analysis since satisfactory
field results were obtained? Further concentrated studying of the
chemical make-up of these sex pheromones with revised and narrowed
biological assays are necessary to prove the existance of only one major
component in these insect species.
Pheromone traps and other synthetically produced Scarab lures are
being produced and used in field testing as a means to control this pest
(Ladd et. al. 1981, Reed et. al. 1991, Cherry and Klein 1992, Potter and
Haynes 1993, Hasegawa et. al. 1993). Because of the severe economic and
biological damage this pest can cause, continued measures of control,
using IPM practices, are a necessary component to the future of Scarab
management.
Table 1. Sex Pheromones of the Scarabaeidae: Summary
SP = sex pheromone
MT = methods used in identification process
Anomala cuprea Hope
SP: (R,Z)-5-(-)-(oct-1-enyl)oxacyclopentan-2-one
(R,Z)-5-(-)-(dec-enyl)oxaclyclopentan-2-1
MT: GC, EAD, EI-MS, behavioral bioassay
Anomala daimiana Harold
SP: 2-(E)-nonenol
(R,Z)-5-(-)-(oct-1-enyl)oxacyclopentan-2-one
MT: GC, MS, EAD, behavioral bioassays
Anomala rufocuprea Motschulsky
SP: methly-(Z)-5-tetradecenoate
MT: GC, HPLC, MS, behavioral bioassay
Anomala schonfeldti Ohaus
SP: 2-(E)-nonenol
MT: GC, EAD, FID, behavioral bioassay
Blitopertha orientalis
SP: (Z) and (E)-tetradec-7-en-2-one
MT: GC, MS, behavioral bioassy
Costelytra zealandica White
SP: Phenol
MT: GLC, MS, paper chromatography, measurements of mass to charge
ratios, behavioral bioassay
Holotrichia parallela Motschulsky
SP: L-isoeucine methyl ester (LIME)
R-(-)-linalool
MT: GC, EAD, MS, behavioral bioassay
Popillia japonica Newman
SP: (R,Z)-5-(-)-(dec-1-enyl)oxacyclopentan-2-one
MT: MS, infrared spec., NMR, chemical transformations, behavioral
bioassays
Literature Cited
Baskaran, S., I. Islam, S. Chandraskaran (1990) A general approach to
the synthesis of butanolides: synthesis of the sex pheromone
of the Japanese beetle. J. Org. Chem. 55:891-895.
Borrer, D.J., C.A. Triplehorn, N.F. Johnson (1989) An introduction to
the study of insects, 6th ed. Saunders College Publ.:
Philadelphia, 1989.
Cherry, R.H. and M.G. Klein (1992) Attraction fo adult Euphoria
sepulcharlis (Coleoptera: Scarabaeidae) to aromatic compounds.
Florida Entomol. 75(3):383-385.
Doolittle, R.E., J.H. Tumlinson, A.T. Proveaux, R.R. Heath (1980)
Synthesis of the sex pheromone of the Japanese beetle. J. Chem.
Ecol. 6(2):473-485.
Ebata, T., H. Kawakami, K. Koseki, K. Matsumoto, H. Matsushita (1992)
Synthesis of japonilure, the pheromone of Japanese beetle.
Biosci. Biotech. Biochem. 56(5):818-819.
Hasegawa, M., W.S. Leal, M. Sawada (1993) Field evaluation of
Anomala schonfeldti ohaus (Coleoptera: Scarabaeidae) synthetic
sex pheromone. J. Chem. Ecol. 19(7):1453-1459.
Henzell, R.F., M.D. Lowe (1970) Sex attranctant of the grass grub
beetle. Science 168:1005-1006.
Koseki, K., T. Ebata, T. Kadokura, H. Kawakami, M. Ono, H. Matsushita
(1993) Synthesis of sex pheromones of the Japanese beetle and
cupreus chafer beetle. Tetrahedron 49(27):5961-5968.
Ladd, T.L., M.G. Klein, J.H. Tumlinson (1981) Phenethyl propionate +
eugenol + geraniol (3:7:3) amd japonilure: a highly effective
joint lure for Japanese beetles. J. Econ. Entomol. 74:665-667.
Leal, W.S., F. Mochizuki, S. Wakamura, T. Yasuda (1992)
Electroantennographic detection of Anomala cuprea Hope
(Coleoptera: Scarabaeidae) sex pheromones. Appl. Entomol. Zool.
27(2):289-291.
Leal, W.S.,M. Sawada, S. Matsuyama, Y. Kuwahara, M. Hasegwa (1993)
Unusual periodicity of sex pheromone production in the large
black chafer Holotrichia parallela. J. Chem. Ecol. 19(7):1381-
1391.
Leal, W.S., M. Hasegawa, M. Sawada (1992) Identification of Anomala
schonfeldti sex pheromone by high resolution GC-behavior
bioassay. Naturwissenschaften 79:518-519.
Leal, W.S., M. Hasegawa, F. Mochizuki, T. Yasuda (1992) Behavioral
and electrophysiological evidence of sex pheromone(s) in
Anomala schonfeldti Ohaus (Coleoptera: Scarabaeidae). Appl.
Entomol. Zool. 27(4):592-594.
Leal, W.S., M. Sawada, M. Hasegwa (1993) The Scarab beetle Anomala
daimiana utilizes a blend of two other Anomala spp. sex
pheromones. Naturwissenschaften 80:181-183.
Leal, W.S. (1991) (R,Z,)-5-(-)-(Oct-1-enyl)oxacyclopentan-2-one, the
sex pheromone of the Scarab beetleAnomala cuprea.
Naturwissenschaften 78:521-523.
Leal, W.S. (1993) (Z)- and (E)-tetradec-7-en-2-one, a new type of
sex pheromone from the Oriental beetle. Naturwissenschaften
80:86-87.
Leal, W.S., F. Mochizuki (1993) Sex pheromone reception in the Scarab
beetle Anomala cuprea, enantiomeric discrimination by sensilla
placodea. Naturwissenschaften 80:278-281.
Leal, W.S., M. Sawada, M. Hasegawa (1993) The Scarab beetle
Anomala cuprea utilizes the sex pheromone of Popillia
japonica as a minor component. J. Chem. Ecol. 19(7):1303- 1313.
Leal, W.S., S. Matsuyama, Y. Kuwahara, S. Wakamura, M. Hasegwa (1992)
An amino acid derivative as the sex pheromone of a Scarab
beetle. Naturwissenschaften 79:184-185.
Mayer, M. S. and J.R. Mclaughlin (1991) Handbook of insect pheromones
and sex attractants. CRC Press: Boca Raton, 1991.
Potter, D.A. , K.F. Haynes (1993) Field testing pheromone traps for
predicting masked chafer (Coleoptera: Scarabaeidae) grub
density in golf course turf and home lawns. J. Entomol. Sci.
28(2):205-212.
Reed, D.K., M.H. Lee, M.G. Klein (1991) Attraction of Scarab beetle
populations (Coleoptera: Scarabaeidae) to Japanese beetle lures
in the Republic of Korea. Agric. Ecosystems Environ. 36:163-174.
Tumlinson, J.H., M.G. Klein, R.E. Doolittle, T.L. Ladd, A.T. Proveaux
(1977) Identification of the female Japanese beetle sex
pheromone: inhibition of male response by an enantiomer.
Science 197: 789-792.