Male-produced aggregation pheromones were demonstrated in Carpophilus hemipterus (L.), Carpophilus lugubris Murray, and Carpophilus freemani Dobson (Coleoptera: Nitidulidae) using a wind-tunnel bioassay. The attractiveness of the pheromones is greatly enhanced by volatiles from a host plant, and combinations of pheromone and food volatiles typically attract 3-10 times more beetles than either source by itself. The pheromones consist of a series of 12-, 13-, 14-, and 15-carbon unsaturated hydrocarbons. The most abundant of these in C. hemipterus is (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene. In C. lugubris, the most abundant is (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, and in C. freemani, (2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to aggregation pheromones of insects, particularly 
the nitidulid species Carpophilus hemipterus, C. lugubris, and C. 
freemani, and the use of these pheromones in combination with host plant 
volatiles to aid in insect control as, for example, in pheromone-baited 
traps. 
2. References 
Throughout this application, various publications are referenced by the 
name of the author and date of publication within parentheses. Full 
citations for these references may be found at the end of the 
specification, listed in alphabetical order. 
3. Description of the Prior Art 
Insect-produced volatiles (e.g., pheromones) and host plant odors (e.g., 
kairomones) may facilitate location of conspecifics for mating and 
orientation to acceptable host plants for feeding and oviposition. It is 
known that in several, but not all, insect species (e.g., bark beetles) 
pheromones and a few specific plant odors, such as monoterpenes, may act 
in synergy, each enhancing the attraction of the other (Borden, 1984). 
Carpophilus hemipterus (L.) (Coleoptera: Nltidulidae) is a worldwide pest 
attacking agricultural commodities such as ripe and dried fruit, corn, 
wheat, oats, rice, beans, nuts, peanuts, cotton seed, copra, spices, 
sugar, honey, and other materials (Hinton, 1945). It is also able to 
vector microorganisms responsible for the souring of figs (Hinton, 1945) 
and fungi which contaminate corn and produce mycotoxins (Wicklow et al., 
1988). 
The dusky sap beetle, Carpophilus lugubris Murray (Coleoptera: Nitidulidae) 
is distributed from Brazil through Central America (Parsons, 1943) and 
probably throughout the United States (Sanford, 1958). It is found in ripe 
and decomposing fruit and vegetables (Sanford and Luckman, 1963), trees 
infected with oak wilt (Dorsey et al., 1953; Norris, 1953), and poultry 
manure (Pfeiffer and Axtell, 1980). It is probably most important as a 
pest of sweet corn (Connell, 1956; Sanford, 1958; Connell, 1975; Tamaki et 
al., 1982), and can cause large amounts of corn to be rejected at 
canneries (Luckman and Hibbs, 1959). In addition, it appears to be a 
vector of oak wilt (Dorsey et al., 1953; Norris, 1953; Appel, 1986), and 
mycotoxin-producing fungi that contaminate corn (Wicklow et al., 1988). 
Although tight-husked corn can provide some control, this may be defeated 
when corn earworms or other insects provide entry holes (Connell, 1956; 
Tamaki et al., 1982). However, in many cases these insects are able to 
enter the ears without assistance (Connell, 1956; Tamaki et al., 1982). 
The loose-husked varieties of dent (field) corn adopted in association 
with the use of mechanical harvesting promote ready entry sites for these 
insects (Connell, 1956). 
Carpophilus freemani Dobson infests sweet corn (Sanford and Luckman, 1963) 
and corn seed and corn meal (Connell, 1975). It is a principal pest of 
figs (Smilanick and Ehler, 1976) and the principal vector of Ceratocystis 
canker of stone fruits including almonds, prunes, peaches, and apricots 
(Moller et al., 1969). 
Field traps have been used to monitor or attempt to control these and other 
nitidulid species, and much research has gone into trap baits. Fermenting 
fig paste has been used as a trap bait for C. hemipterus (Obenauf et al., 
1976}. Smilanick et al. (1978) determined that a 1:1:1 mixture of 
acetaldehyde, ethyl acetate, and ethanol was an even more effective bait 
for C. hemipterus than fig paste, but trap catches were still relatively 
small, given the huge beetle populations. Due to the low activity of 16 
other host volatiles tested, Smilanick et al. (1978) concluded that C. 
hemipterus "appears to use a restricted number of olfactory stimuli to 
locate suitable hosts." Previously reported methods of monitoring C. 
lugubris have been of limited effectiveness. It is well known that these 
insects can be attracted by fermenting baits (Luckman and Hibbs, 1959). 
Specific methods include using freshly sawn oak or maple blocks in 
combination with vinegar and fungi (Neel et al., 1967; Dorsey and leach, 
1956). However, the attractiveness of these baits varies over time due to 
changes in fermentative activity (Neel et al., 1967). Previously reported 
methods of attracting C. freemani are also of limited effectiveness. The 
only reported method specifically describing C. freemani attraction is 
that of Smilanick et al. (1978). The response of C. freemani to 
Smilanick's 3-component mixture appeared to be relatively poor compared to 
that of C. hemipterus, and not significantly different from fig paste or 
controls. Alm et al. (1985, 1986) demonstrated that esters such as propyl 
propionate and butyl acetate were effective baits for Glischrochilus 
quadrisignatus, another economically important nitidulid, but did not 
compete with banana. In nature, these chemicals exist in the host plant, 
are produced by microorganisms which have established on the plants, or 
both. Curiously, no pheromones have teen reported for nitidulid beetles, 
even though attractants of this type would probably be potent trap baits 
or additives to presently used baits. Pheromones have teen reported for a 
large number of other beetle species. 
SUMMARY OF THE INVENTION 
We have now surprisingly found that male-produced aggregation pheromones 
are secreted by C. hemipterus, C. lugubris and C. freemani. The 
attractiveness of the pheromone complexes is greatly enhanced by a range 
of volatiles from a host food source. 
It is an object of this invention to describe the isolation and synthesis 
of the hydrocarbon components of the aggregation pheromones. 
Another object of the invention is to teach an improved method of 
attracting insects by the combined use of aggregation pheromones and food 
volatiles. 
Other objects and advantages of the invention will become readily apparent 
from the ensuing description. 
DETAILED DESCRIPTION OF THE INVENTION 
We have now discovered that male C. hemipterus, C. lugubris, and C. 
freemani beetles produce volatile hydrocarbon mixtures which are 
attractive to both sexes and are, therefore, termed aggregation 
pheromones. The pheromone complexes are especially effective when used in 
combination with volatiles from a food source. The isolation, 
identification, and synthesis of the pheromones and their biological 
activity, alone and in conjunction with food volatiles, are described 
below. 
It is understood that host food source volatiles may be produced directly 
by the host plant, by microorganisms such as yeasts which are growing on 
plant tissues, or by both. 
The hydrocarbons of this invention may be represented by the general 
formula: 
##STR1## 
wherein R.sup.1 and R.sup.2 are independently selected from hydrogen or 
lower alkyl, and n is zero or one. 
The natural and synthetic compounds used in this work are listed below with 
assigned numbers, which are used in the following text and tables. 
Structures of the compounds are shown in Table I. 
TABLE I 
______________________________________ 
Synthetic Hydrocarbons 
______________________________________ 
##STR2## 1 
##STR3## 2 
##STR4## 3 
##STR5## 4 
##STR6## 5 
##STR7## 6 
##STR8## 7 
##STR9## 8 
##STR10## 9 
##STR11## 10 
##STR12## 11 
##STR13## 12 
##STR14## 13 
##STR15## 14 
______________________________________ 
______________________________________ 
Num- 
Compound ber 
______________________________________ 
Compounds found in beetles: 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-3,5,7-Trimethyl-2,4,6,8- 
decatetraene 1 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-3,5,7-Trimethyl-2,4,6,8- 
undecatetraene 2 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-7-Ethyl-3,5-dimethyl-2,4 
,6,8-decatetraene 3 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-5-Ethyl-3,7-dimethyl-2,4 
,6,8-decatetraene 4 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-4,6,8-Trimethyl-2,4,6,8- 
undecatetraene 5 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-7-Ethyl-3,5-dimethyl-2,4 
,6,8- 6 
undecatetraene 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-5-Ethyl-3,7-dimethyl-2,4 
,6,8- 7 
undecatetraene 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E)-5-Ethyl-3-methyl-2,4,6-nonatriene 
14 
Compounds not found in beetles: 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- Z,8 .sub.-- E)-3,5,7-Trimethyl-2,4,6,8- 
decatetraene 8 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- Z,8 .sub.-- E)-3,5,7-Trimethyl-2,4,6,8- 
undecatetraene 9 
(4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-2,3,5,7-Tetramethyl-2,4,6,8-decatetr 
aene 10 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-3,5,7-Trimethyl-2,4,6,8- 
dodecatetraene 11 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-3,5,7,9-Tetramethyl-2,4, 
6,8-undecatetraene 12 
(2 .sub.-- E,4 .sub.-- E,6 .sub.-- E,8 .sub.-- E)-5,7-Diethyl-3-methyl-2,4 
,6,8-decatetraene 13 
______________________________________ 
SYNTHESIS OF HYDROCARBONS 
Compounds 1-14 were prepared as model compounds to aid in structure 
identification of the natural pheromones and as test materials for the 
bioassay. Synthetic reactions were performed as described in the 
literature for similar systems. All reactions, except for the formation of 
phosphonium salts, were monitored by GC and mass spectrometry. 
Intermediates were generally used in the subsequent reactions without 
purification, other than drying over sodium sulfate and removal of 
solvent. The final step in the synthesis of each tetraene was a Wittig 
reaction between an aldehyde (Compounds 15-20, Table II) and a phosphonium 
salt (Compounds 21-27, Table III). 
TABLE II 
__________________________________________________________________________ 
Aldehyde Intermediates 
__________________________________________________________________________ 
##STR16## 
##STR17## 
##STR18## 
__________________________________________________________________________ 
TABLE III 
__________________________________________________________________________ 
Phosphonium Salt Intermediates 
__________________________________________________________________________ 
##STR19## 
##STR20## 
##STR21## 
##STR22## 
__________________________________________________________________________ 
In the synthesis of the aldehyde intermediates (Table II), three 
commercially available, carbonyl starting materials were used: 
2-methyl-2(E)-butenal, 2-methyl-2(E)-pentenal, and acetone. These and 
subsequent product aldehydes were subjected to two synthetic schemes. In 
scheme A, the carbonyl starting material was coupled with triethyl 
2-phosphonopropionate in a Wittig-Horner condensation, forming an ethyl 
ester (Gallagher and Webb, 1974). This reaction produces E double bonds 
stereoselectively (Boutagy and Thomas, 1974). The ester functional group 
was then reduced to the corresponding alcohol with LiAlH.sub.4 as 
described by Mori (1976) for another ethyl ester, and the alcohol was 
oxidized with periodinane (Dess and Martin, 1983) to the corresponding 
aldehyde. These reactions proceeded cleanly, and by capillary GC a single 
compound usually accounted for over 90% of the volatile reaction products. 
Scheme B was exactly the same as scheme A except that triethyl 
2-phosphonobutyrate was used in the Wittig-Horner reaction. This allowed 
incorporation of an ethyl side chain into the product instead of a methyl 
group. 
To make the phosphonium salts (Table III), six commercially available 
starting materials were used: bromoethane, 1-iodopropane, 1-bromobutane, 
2(E)-butenal, 2(E)-pentenal, and 2-butanone. These and subsequent 
intermediates were subjected to three synthetic schemes. 
Scheme C was exactly like scheme A except that triethyl 2-phosphonoacetate 
was used in the Wittig-Horner reaction. This reaction, which was used to 
link a primary phosphonate anion with a ketone, produced the E and Z 
isomers in a 60:40 ratio. Fortunately, the desired final tetraene product 
elaborated from the E intermediate was easily separated 
chromatographically (AgNO.sub.3 -HPLC) from the other isomer. 
In scheme D, the alkyl halide was refluxed with triphenylphosphene in 
acetonitrile for 8 hr, followed by removal of solvent and crystallization 
of the phosphonium salt trituration under dry ether. 
In scheme E, the aldehyde starting material was alkylated with 
methylmagnesium bromide (Brooks and Snyder, 1955; except that a 
commercially prepared Grignard reagent was used). Then the alcohol was 
converted to the bromide with PBr.sub.3 (Noller and Dinsmore, 1943; except 
that the bromide was recovered by extraction with hexane rather than by 
distillation). Finally, the secondary, allylic bromide was converted to 
the phosphonium salt as described for scheme D. 
Some allylic rearrangement occurred as the unsaturated salts were formed. 
Based on analysis of subsequent reaction products, salt 26 represented 
about 10% of the mixture of 25 and 26. Rearrangement of 24 was not a 
problem because of symmetry; and allylic rearrangement of 27, if it 
occurred, was not a problem because the resulting tertiary salt could not 
take part in a Wittig reaction. 
The aldehyde and phosphonium salt intermediates were coupled in a final 
Wittig reaction as described by Sonnet (1974) to form the desired 
hydrocarbons (Table IV). With unsaturated phosphonium salts (24-27), the 
reaction formed both the E and Z isomers of the final double bond in 
approximately equal proportions. However, with the saturated salts 
(21-23), the Z isomer was always a minor product (ca. 10%. 
The synthetic tetraenes were purified first by column chromatography on 
silica (hexane as solvent, Example 4). There appeared to be some 
decomposition on this column (formation of yellow color, which remained on 
the column), but the expected products were always recovered. Second, the 
geometrical isomers of the tetraenes were separated by HPLC on the 
silver-nitrate column (Example 4, 25% or 10% toluene in hexane). It was 
usually possible to obtain geometrical isomers from this column which, by 
GC, were not contaminated by the other isomer. Two tetraenes with Z double 
bonds (8 and 9), were included in bioassay and analytical studies for 
comparison; but the insect-derived tetraenes were found to have only E 
double bonds. 
TABLE IV 
______________________________________ 
Synthetic Hydrocarbons 
______________________________________ 
##STR23## 1 
##STR24## 2 
##STR25## 3 
##STR26## 4 
##STR27## 5 
##STR28## 6 
##STR29## 7 
##STR30## 8 
##STR31## 9 
##STR32## 10 
##STR33## 11 
##STR34## 
##STR35## 12 
##STR36## 13 
##STR37## 14 
______________________________________ 
Carpophilus hemipterus 
The isolation, identification, synthesis, and biological activity of the 
aggregation pheromones of C. hemipterus are described below. 
The C. hemipterus beetles were reared on a pinto bean diet as described by 
Dowd (1987). The isolation of the pheromone for this species was guided by 
a wind-tunnel bioassay (described in detail in Example 1). Briefly, the 
wind tunnel usually contained 200-400 beetles of mixed sex. Bioassay tests 
always included two treatments, placed side by side, in the upwind end of 
the wind tunnel. The beetles located bait materials by flying upwind to 
the source of the attractive volatiles. The number of beetles landing at 
each bait was used as a measure of its attractiveness. Simultaneous 
testing of two treatments made precise comparisons possible, without 
having to control the numbers of beetles in the wind tunnel or their 
activity level too closely. 
We believe the wind-tunnel bioassay to be more ecologically relevant than 
the more classical "pit-fall" bioassay used for many stored-product 
beetles (see Phillips and Burkholder, 1981). The beetles are excellent 
fliers and, presumably, find and colonize new host sites in the field 
through flight activity. 
One necessary condition for a successful bioassay was that the beetles in 
the wind tunnel be starved for a number of hours before tests were 
conducted. When beetles were transferred from their food medium into the 
wind tunnel, they would quickly form aggregations in the corners and then 
become motionless. After several hours, a few beetles would begin to move 
about and take flight spontaneously. This dispersal from the aggregations 
became more pronounced with time, and responses to pheromone or food baits 
occurred only after this flight activity had begun. By starving the 
beetles for 16 hours prior to beginning tests, responses occurred rapidly 
enough (&gt;10 per 3-min period) to be useful for monitoring pheromone 
isolation. 
The aggregation pheromone could be obtained from the beetles either by 
extracting whole cultures of the insects (beetles and rearing medium, 
Example 2) or by collecting volatiles from a culture (Example 3). Initial 
experiments and synergism studies were conducted with the whole extracts. 
The pheromone is produced by male beetles. When an extract of a whole 
culture containing only males was tested against an equivalent extract 
derived from females, the beetles in the wind tunnel 1 flew preferentially 
to that from males. The total bioassay counts were 71 and 1, respectively, 
over eight 10-min observation periods (ca. 1 beetle equivalent per test). 
However, both male and female beetles responded readily to the pheromone. 
In one experiment, the beetles attracted to a culture of males were 
captured and sexed; of the 142 beetles which responded, 62 were males and 
80 were females. The remaining data submitted here represent totals over 
both sexes. 
A methylene chloride extract of whole cultures with males was fractionated 
on silicic acid; but qualitatively, each of the five fractions was 
inactive in the bioassay compared with the original extract. However, the 
recombined fractions were again quite attractive, indicating the active 
compounds had eluted from the column but that more than one chemical was 
required for attraction. Collection of volatiles from living beetles 
(Tenax collection) gave similar results. 
It was suspected that both male-derived and diet-derived volatiles were 
responsible for the activity of the whole cultures. To identify which 
fraction of the male-derived extract contained the pheromone, we tested 
combinations of the five chromatographic fractions (described in Example 
4). In each combination, one of the fractions was derived from cultures 
with only males and the remaining four from cultures with only females. 
Each combination was tested against the whole extract of the female 
culture (the control in this experiment). Thus, all the bioassay 
treatments would contain the full complement of diet compounds as well as 
any "general" metabolites produced by beetles of both sexes and compounds 
from any associated microorganisms. The attractiveness of the combination 
of fractions would be expected to differ from the control only if the 
single male-derived fraction contained the pheromone. From Table V, it is 
clear that the hexane fraction was the primary source of male-specific 
attractants. The pheromone was quite nonpolar, indicative of hydrocarbons. 
Furthermore, only one male-derived fraction was required for potent 
pheromonal activity. Thus the pheromone appeared not to include components 
of widely different polarity. 
The male-derived attractant was synergized by a wide variety of host 
volatiles besides those from the rearing medium (Table VI). Effective 
coattractants included crude plant materials, various yeast cultures, 
single chemicals (especially esters), and mixtures of chemicals. It is 
noteworthy that the best previously reported attractant (ethanol, ethyl 
acetate, and acetaldehyde) attracted over three times more beetles when 
the pheromone was added to it. Because reproduction in these beetles 
occurs at feeding sites, the enhanced attraction to combined host- and 
beetle-derived volatiles is undoubtedly of great ecological importance. 
Effective chemical synergists include: (1) C.sub.1 -C.sub.4 straight and 
branched alcohols; C.sub.5 and greater straight chain alcohols. (2) 
C.sub.2 and greater straight and branched acids (except for those which 
had two methyl groups in the 2 position of the structure). (3) A large 
number of esters, including: (a) all esters in which both the alcohol and 
acid moieties are unbranched, especially wherein said moieties are within 
the range of C.sub.1 -C.sub.8, except for methyl formate, (b) methyl 
esters with branched acid moieties having no more than 1 methyl branch, 
and (c) 1-methylethyl acetate and 2-methylpropyl acetate. (4) Aldehydes, 
ketones, and water. The only bifunctional sample tested, 
2-hydroxypropanoic acid, was effective; this more complex compounds could 
also be synergistic. 
TABLE V 
______________________________________ 
Activity of Silica Fractions of 
Male-Derived Extract in Wind Tunnel 
Mean Biossay Count (n = 6) 
Male-Derived Fraction 
Fraction Combination.sup.a 
Control.sup.b 
______________________________________ 
Hexane 23.3* 0.5 
5% Ether-hexane 
1.5 0.5 
10% Ether-hexane 
1.5 1.8 
50% Ether-hexane 
1.2 0.8 
10% MeOH--CH.sub.2 Cl.sub.2 
2.2 1.3 
______________________________________ 
.sup.a Each malederived fraction was combined with the four complementary 
fractions derived from females. The only significantly active combination 
is marked with an (*). 
.sup.b The control for this experiment was the whole extract of a culture 
of females. Therefore, each bait in the experiment contained all the same 
dietderived compounds, as well as any compounds shared by both sexes of 
beetles. 
TABLE VI 
__________________________________________________________________________ 
Synergistic Interactions Between Host Plant 
Volatiles and Pheromones of Carpophilus hemipterus 
Mean bioassay count (n&gt; = 8) 
Volatile Volatile 
Pheromone 
Volatile + Pheromone 
__________________________________________________________________________ 
Crude Host Materials 
Orange juice 0.5 a 
1.0 a 10.6 b 
Apple juice 1.2 a 
1.3 a 15.6 b 
Juice of corn kernels 
0.1 a 
2.4 b 6.3 c 
Corn silk 0.0 a 
4.5 b 8.8 c 
Corn husk 0.0 a 
2.8 b 8.2 c 
Corn kernel 0.0 a 
2.5 b 13.2 c 
Corn kernel + silk 
0.5 a 
3.0 b 21.0 c 
Baker's yeast on agar medium 
0.4 a 
1.9 a 6.3 b 
Baker's yeast on banana 
2.2 a 
3.5 a 30.4 b 
Z.b. on banana 3.2 a 
3.0 a 24.8 b 
Esters 
Methyl formate 1.7 a 
16.2 b 
18.9 b 
Methyl acetate 1.5 a 
6.1 b 21.3 c 
Methyl propanoate 
6.4 a 
6.4 a 43.5 b 
Methyl butanoate 8.6 a 
3.1 b 41.7 c 
Methyl 2-methylpropanoate 
0.9 a 
2.4 b 15.5 c 
Methyl pentanoate 
1.2 a 
6.1 b 25.8 c 
Methyl 2-methylbutanoate 
2.1 a 
6.5 b 30.4 c 
Methyl 3-methylbutanoate 
0.0 a 
8.1 b 13.5 c 
Methyl 2,2-dimethylpropanoate 
0.3 a 
2.9 b 4.5 b 
Methyl 4-methylpentanoate 
0.7 a 
6.5 b 21.5 c 
Ethyl acetate 0.3 a 
2.9 b 14.7 c 
Ethyl propanoate 1.4 a 
5.2 b 35.9 c 
Ethyl butanoate 0.3 a 
2.4 b 18.7 c 
Ethyl 2-methylpropanoate 
0.1 a 
1.3 b 3.2 b 
Ethyl 3-methylbutanoate 
0.3 a 
5.0 b 4.0 b 
Propyl acetate 1.0 a 
2.6 a 17.2 b 
1-Methylethyl acetate 
1.4 a 
7.0 b 25.4 c 
Propyl propanoate 
2.5 a 
2.5 a 72.0 b 
Butyl acetate 0.1 a 
2.7 b 10.6 c 
2-Methylpropyl acetate 
0.0 a 
2.4 b 8.6 c 
1-Methylpropyl acetate 
0.2 a 
2.3 b 3.6 b 
1,1-Dimethylethyl acetate 
0.0 a 
2.4 b 1.2 b 
Butyl propanoate 0.0 a 
10.0 b 
34.0 c 
Pentyl acetate 1.7 a 
3.0 a 14.3 b 
1-Methylbutyl acetate 
0.9 a 
6.0 b 9.4 b 
2-Methylbutyl acetate 
0.0 a 
5.3 b 2.7 b 
3-Methylbutyl acetate 
0.4 a 
5.2 b 6.4 b 
1-Ethylpropyl acetate 
0.3 a 
6.0 b 3.8 b 
Heptyl hexanoate 0.4 a 
1.0 b 8.3 c 
Octyl acetate 0.4 a 
2.6 b 13.9 c 
Benzyl acetate 0.1 a 
0.7 ab 
1.8 b 
Alcohols 
Methanol 3.1 a 
1.9 a 13.9 b 
Ethanol 1.3 a 
3.1 a 16.3 b 
1-Propanol 4.4 a 
14.9 b 
61.0 c 
2-Propanol 6.6 a 
8.7 a 41.0 b 
1-Butanol 0.1 a 
2.6 b 8.0 c 
2-Methyl-1-propanol 
0.2 a 
33.8 b 
24.5 b 
1,1-Dimethylethanol 
1.5 a 
7.8 b 32.9 c 
2-Butanol 0.4 a 
7.1 b 17.8 c 
2-Methyl-1-butanol 
0.2 a 
4.7 b 3.3 b 
3-Methyl-1-butanol 
1.2 a 
15.8 b 
11.5 b 
1-Heptanol 0.2 a 
1.6 b 5.5 c 
Acids 
Formic acid 0.1 a 
1.2 b 1.7 b 
Acetic acid 0.3 a 
3.0 b 8.7 c 
Propanoic acid 6.7 a 
8.9 a 74.4 b 
2-Hydroxypropanoic acid 
0.6 a 
2.5 a 8.7 b 
Butanoic acid 0.4 a 
3.0 b 19.3 c 
2-Methylpropanoic acid 
0.2 a 
10.2 b 
7.1 b 
Pentanoic acid 1.5 a 
5.9 b 25.8 c 
2-Methylbutanoic acid 
4.3 a 
3.6 a 34.1 b 
3-Methylbutanoic acid 
0.9 a 
7.2 b 21.0 c 
2,2-Dimethylpropanoic acid 
0.1 a 
6.6 b 6.4 b 
3-Methylpentanoic acid 
3.0 a 
5.8 b 30.0 c 
4-Methylpentanoic acid 
2.7 a 
3.7 b 24.2 c 
2,2-Dimethylbutanoic acid 
0.1 a 
11.4 b 
15.6 b 
Other Single Components 
Acetaldehyde 0.0 a 
2.4 b 7.8 c 
Propanal 2.4 a 
0.8 b 7.0 c 
2-Pentanone 0.2 a 
0.8 a 7.7 b 
Water 0.2 a 
6.1 b 10.4 c 
Mixtures (all 1:1:1) 
Ethanol:acetaldehyde: 
9.2 a 
4.5 b 29.6 c 
ethyl acetate 
Ethanol:ethyl butanoate: 
2.1 a 
2.2 a 32.4 b 
2-hydroxypropanoic acid 
Ethanol:ethyl propanoate: 
8.9 a 
5.0 b 43.9 c 
propanoic acid 
Ethanol:ethyl propanoate: 
6.6 a 
3.9 b 28.2 c 
acetaldehyde 
Ethanol:ethyl 2-methylpropanoate: 
2.0 a 
5.3 b 16.1 c 
2-hydroxypropanoic acid 
__________________________________________________________________________ 
Each line represents one experiment; data are mean counts per 3min test. 
In each line, means followed by the same letter are not significantly 
different (LSD, P = 0.05). Baker's yeast = Saccharomyces cerevisiae; the 
agar medium was potato dextrose agar. Z.b. = Zygosaccharomyces bailii. Th 
pheromone source was the hydrocarbon fraction of an extract derived from 
whole culture containing male beetles; the concentration was adjusted so 
that there was 0.5-1.0 ng of the major pheromone component per test; in 
each line of the table the amount of pheromone used was constant. 
The active compound from the male beetles appeared to have at least one 
double bond, because the 10% ether-hexane fraction from the AgNO.sub.3 
column contained most of the activity (Table VII). A hydrocarbon without 
double bonds would have eluted with hexane. Further purification by HPLC 
with the size-exclusion column yielded two consecutive 1-ml fractions that 
were quite active (Table VII). The size-exclusion column was very valuable 
for separating inert hydrocarbons of high molecular weight from the 
attractants. Male-derived Tenax collections also provided active 
hydrocarbons, and these were fractionated by HPLC on the AgNO.sub.3 
column. Four consecutive 0.5-ml fractions had activity (Table VII). As 
with the open column, the retention of active fractions indicated 
unsaturation in the pheromone. 
Parallel chromatographic fractions derived from female beetles were 
prepared, and the fractions from both sexes were analyzed by GC. In the 
active, male-derived HPLC fractions there were at least 11 compounds that 
were absent from the females (Table VIII). Considering both the GC and 
bioassay data, it was clear that no single compound was absolutely 
required for activity and that more than one subset of male-specific 
hydrocarbons was sufficient to elicit attraction. However, complete 
separation of these compounds was not obtained by any HPLC method. 
Preparative GC did not provide pure compounds either, because many were 
too similar in GC retention and too labile to survive this technique. 
In the extracts of male cultures, 1 beetle equivalent contained 
approximately 1 ng of the major component (I=13.83, Table VIII). In a 
typical Tenax collection, 1 beetle-day represented ca. 0.5 ng of this 
component. Because the beetles could live for several months in the 
aeration flasks, the Tenax collections were the richer source of active 
hydrocarbons and, furthermore, these were relatively easy to purify. 
TABLE VII 
______________________________________ 
Activity of Chromatographic Fractions Derived 
from Male C. hemipterus Hydrocarbons.sup.a 
Mean Bioassay Count (n = 4) 
Fraction Description 
Fraction + Coattractant.sup.b 
Coattractant.sup.b 
______________________________________ 
AgNO.sub.3 fractions (open column, from culture extract) 
Hexane 1.0 1.3 
5% Ether-hexane 
15.0* 2.0 
10% Ether-hexane 
33.3* 1.3 
25% Ether-hexane 
6.7 2.5 
Ether (first) 
1.3 2.0 
Ether (second) 
0.8 1.0 
Size-exclusion fractions (HPLC, from AgNO.sub.3 10% 
ether-hexane fraction, above) 
8-10 ml after injection 
0.8 1.0 
10-11 ml 12.0* 1.5 
11-12 ml 9.3* 1.0 
12-13 ml 3.0 1.0 
13-14 ml 1.0 1.8 
14-15 ml 1.0 1.3 
15-16 ml 1.5 1.0 
AgNO.sub.3 fractions (HPLC, from Tenax collections) 
3,0-4.5 ml after 
0.0 0.3 
injection 
4.5-5.0 ml 0.0 0.0 
5.0-5.5 ml 0.5 0.0 
5.5-6.0 ml 12.8* 0.3 
6.0-6.5 ml 12.0* 0.0 
6.5-7.0 ml 25.8* 0.8 
7.0-7.5 ml 4.8* 0.3 
7.5-8.0 ml 0.8 0.3 
______________________________________ 
.sup.a Hydrocarbons were isolated by column chromatography on silica prio 
to separations listed in Table. The symbol "*" denotes a statistically 
significant (P &lt; 0.05) difference from the control. 
.sup.b In first two data sets, coattractant was the extract from female 
beetles + diet; in the last experiment, coattractant was propyl acetate 
(10% in mineral oil, 10 .mu.l per test.) 
TABLE VIII 
__________________________________________________________________________ 
Male-Specific Hydrocarbons in C. hemipterus 
HPLC Retention (ml).sup.b 
Retention 
Relative 
Molecular Structure 
Index (I).sup.a 
Amount 
Weight 
Size Exclusion 
AgNO.sub.3 
No. 
__________________________________________________________________________ 
12.44 3% 176 (not detected) 
.sup. 6.0-6.5*.sup.c 
-- 
.sup. 13.08.sup.d 
11% 176 10.5-11.5* 
6.0-6.5* 
-- 
.sup. 13.29.sup.d 
4% 176 11.0-12.0* 
5.0-5.5 
-- 
13.83 57% 176 11.0-12.0* 
6.5-7.5* 
1 
14.22 4% 190 10.0-11.0* 
5.5-6.5* 
4 
14.28 3% 190 10.5-11.5* 
5.5-6.5* 
3 
14.63 7% 190 10.5-11.5* 
6.5-7.0* 
5 
14.76 8% 190 11.0-12.0* 
6.0-7.0* 
2 
14.91 1% 204 10.0-11.0* 
6.0-6.5* 
-- 
15.13 0.4% 204 (not detected) 
5.5-6.0* 
7 
15.15 2% 204 10.0-11.0* 
5.5-6.0* 
6 
__________________________________________________________________________ 
.sup.a Retention index relative to nalkanes; determined from temperature 
programmed runs (10.degree./min) by linear interpolation. 
.sup.b Based on examination of fractions by GC. Many retention volumes 
represent two consecutive HPLC fractions which both contained the 
compound. 
.sup.c *indicates that HPLC fraction was active in bioassay. 
.sup.d Also appears in every fraction where the major hydrocarbon (I = 
13.83) occurs; these may be decomposition products. 
Mass spectra of the unknown compounds were obtained (Example 5). The EI 
mass spectrum of the most abundant compound suggested the molecular weight 
to be 176. This was confirmed by the CI mass spectrum, in which the major 
peaks were 177 (M+1) and 233 (M+57, due to the isobutane reagent gas). The 
molecular weight is consistent with the molecular formula C.sub.13 
H.sub.20, indicating four double-bond equivalents. There was no evidence 
for oxygen or other heteroatoms in the mass spectrum. All fragment ions 
had reasonable C.sub.X H.sub.Y formulae, and the chromatographic evidence 
favored a hydrocarbon also. The other male-specific peaks had similar mass 
spectra, indicating hydrocarbons of 13, 14, or 15 carbons, all with four 
double-bond equivalents (Table VIII). Based on hydrogenation studies, mass 
spectra, ultraviolet spectra, and NMR spectra, it was evident that the 
most abundant pheromone component of C. hemipterus (I=13.83) was structure 
1. This structure was confirmed by synthesis. Two synthetic methods were 
used so that there was no ambiguity about the configurations of the double 
bonds. (All double bonds were either present in the geometrically pure 
starting materials or were formed stereoselectively by known reactions). 
The synthetic compound matched the natural pheromone component in all 
respects: NMR spectrum, mass spectrum, mass spectra of hydrogenation 
products, UV spectrum, HPLC retentions on size-exclusion and AgNO.sub.3 
columns, and GC retentions of hydrogenation products. 
Six minor components present in the male beetles (compounds 2-7) were 
identified by the preparation of model compounds which matched the natural 
compounds exactly. Synthetic targets were chosen based on chromatographic 
retentions and mass spectral fragmentation patterns of the natural 
compounds and their hydrogenated derivatives. Synthetic compounds were 
produced which were identical to the natural ones in GC and HPLC 
retentions, mass spectra, mass spectra of hydrogenated derivatives, and GC 
retentions of hydrogenated derivatives. The synthetic compounds (1-14) 
differed substantially in chromatographic and spectral properties; thus 
the analytical methods used were sufficiently sensitive to discriminate 
among these similar structures. There was not enough of any minor 
component to obtain an NMR spectrum, nor could pure 7 be isolated from the 
beetles in large enough quantities for hydrogenation to be possible. NMR 
spectra were obtained for the synthetic compounds to confirm that the 
target structures were indeed produced. 
A mixture of the tetraenes was prepared to mimic the natural pheromone as 
closely as possible (Table IX). This blend was comparable in activity to 
the natural pheromone (Table X), and both treatments were very active 
compared with the control. In addition to the identified tetraenes, the 
beetle-derived collection contained (by GC) low levels of still 
unidentified tetraenes and compounds derived from the beetle diet that 
were not separated from the active constituents during sample preparation. 
From the bioassay data, these additional compounds appeared to be 
biologically inert. Therefore, the mixture of the synthesized tetraenes, 
or a subset of these, was sufficient to account for the activity of the 
pheromone. 
Individually, and at the same doses as in the whole mixture, only two of 
the seven synthetic tetraenes were significantly above control levels in 
the bioassay (Table XI). These were the C.sub.14. component, 
(2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene (2), and the C.sub.13 
major component, (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene (1), 
which had 27% and 11% of the activity of the whole mixture, respectively. 
TABLE IX 
______________________________________ 
Composition of Bioassay Mixtures, 
Based on Analysis by GC 
Pheromone Sample 
Structure 
Retention from C. hemipterus 
Synthetic Mixture 
Number Index (GC) 
(pg/10 .mu.l) (pg/10 .mu.l) 
______________________________________ 
1 13.83 1000 1000 
2 14.76 130 135 
3 14.28 80 76 
4 14.22 33 39 
5 14.63 59 64 
6 15.15 19 23 
7 15.13 3 6 
______________________________________ 
TABLE X 
______________________________________ 
Bioassay Comparison of Pheromone Derived 
from C. hemipterus and Synthetic Tetraene Mixture 
Treatment Mean Bioassay Count (n = 24) 
______________________________________ 
Beetle-derived pheromone + propyl 
13.7 a 
acetate 
Synthetic tetraene mixture + 
14.8 a 
propyl acetate 
Propyl acetate (experimental control) 
0.3 b 
______________________________________ 
Beetle-derived and synthetic preparations described in Table III; 10 .mu. 
of each solution used per test. 
TABLE XI 
______________________________________ 
C. hemipterus: Bioassay of Individual Tetraenes 
at the Same Dose as in the Synthetic Mixture 
Structure 
Dose Activity Mean Bioassay Counts (n = 8) 
Number (pg) Index Tetraene 
Synthetic Mix 
Control 
______________________________________ 
2 130 27% 6.9*** 
20.4 1.8 
1 1000 11% 2.0*** 
16.2 0.3 
3 80 4% 1.1 20.5 0.4 
6 19 2% 0.6 20.4 0.2 
4 33 2% 0.7 14.2 0.5 
5 59 -2% 0.2 13.2 0.4 
7 3 -2% 0.5 21.4 0.9 
______________________________________ 
All bioassay treatments, including the control, also contain the 
synergist, propyl acetate (10 .mu.l of a 10% solution in mineral oil). 
Each line represents a balanced incomplete block experiment. Significant 
difference between tetraene and control indicated by *** (P &lt; 0.001). 
Activity index = 100 .times. (tetraene - control)/(synthetic mix - 
control). The activity index expresses the activity of a tetraene as a 
percent of the activity of the synthetic mixture, correcting for controls 
TABLE XII 
______________________________________ 
C. hemipterus: Bioassay of Synthetic Tetraenes, 
All at 1.3 ng Total Tetraene Per Test 
Structure 
Activity Mean Bioassay Count (n = 8) 
Number Index Tetraene Synthetic Mixture 
Control 
______________________________________ 
2 52% 8.9*** 16.2 1.0 
6 47% 5.9*** 12.3 0.3 
3 20% 3.3*** 14.7 0.6 
1 10% 1.9*** 15.4 0.4 
11 7% 1.7** 17.0 0.5 
8 5% 1.4 13.4 0.8 
13 3% 1.6 17.9 1.0 
7 3% 0.9 15.4 0.4 
12 3% 0.8 10.9 0.4 
5 3% 0.8 12.9 0.4 
9 3% 1.8 12.5 1.5 
10 2% 0.8 12.0 0.6 
4 1% 0.5 10.9 0.3 
______________________________________ 
Experimental design and definition of terms as in Table XI. Significant 
differences between tetraenes and controls indicated by ** (P &lt; 0.01) and 
*** (P &lt; 0.001). Again, all treatments contained propyl acetate in 
addition to the compounds indicated. Synthetic mixture (see Table IX) use 
at 1.3 ng per test. 
These components were retested, along with other synthetic tetraenes, at a 
higher dose (1.3 ng test), so that these had the same total ng of tetraene 
as the synthetic mixtures (Table XII). Two more of the individual natural 
tetraenes now showed significant activity: These were the 7-ethyl-C.sub.15 
component (structure 6) and the 7-ethyl-C.sub.14 component (structure 3), 
with 47% and 20% of the activity of the synthetic mixture, respectively. 
The most active C.sub.14 component (structure 2) also showed ca. 2-fold 
increase in activity in this experiment, due to the 10-fold increase in 
dose. Neither of the natural tetraenes with an ethyl group at the 5 
position (structures 4 and 7) was active at any dose, nor was 
(2E,4E,6E,8E)-4,6,8-trimethyl-2,4,6,8-undecatetraene (structure 5). 
Of the tested tetraenes that did not occur in the beetles, only one was 
marginally active. (2E,4E,6E,8E)-3,5,7-Trimethyl-2,4,6,8-dodecatetraene 
(structure 11) was 7% as active as the synthetic mixture, on an equal 
weight basis. 
Thus, by virtue of their relatively high natural amounts and significant 
activity, it appeared that the major C.sub.13 and most active C.sub.14 
components (structures 1 and 2) were the compounds of primary biological 
importance; but two additional compounds, the 7-ethyl-C.sub.14 and 
C.sub.15 components (structures 3 and 6) showed activity when tested at 
20-50 times the original level. A combination of 1 and 2, at the levels 
shown in Table IX, was equivalent in activity to the whole synthetic 
mixture (mean counts were 15.2 and 15.9, respectively, n=24, P=0.50, 
paired t test). 
The four natural components that showed activity alone were tested again, 
in binary combinations, for evidence of synergistic activity (Table XIII). 
The most abundant natural component (1) was used at 1 ng/test; the other 
(2, 3, and 6) were used at 250 pg/test. Counts for three binary mixtures 
were quite low (lines 2, 5, and 6 of Table XIII), despite the observation 
that the beetles in the wind tunnel had responded readily to the standard 
synthetic mixture before and after the experiments were conducted. On the 
other hand, the binary combinations 1+2, 1+6, and 2+3 provided good 
counts consistently. When all six binary mixtures were tested against the 
whole synthetic mixture and the control, only the mixture 1+3 performed 
poorly (Table XIV). 
TABLE XIII 
______________________________________ 
C. hemipterus: Bioassay Activity of 
Six Binary Mixtures of Tetraenes and the Individual 
Components - Mean Bioassay Counts (n = 6) 
Individual 
Components (by Structure No.) 
Control 
1 2 3 6 Binary Mixture 
______________________________________ 
0.7 d 2.5 c 9.1 b -- -- 15.5 a 
0.6 b 5.2 a -- 1.1 b -- 3.4 a 
1.3 c .sup. 2.2 bc 
-- -- 3.4 b 11.1 a 
0.8 c -- 11.1 a 4.0 b -- 14.4 a 
0.4 b -- 2.7 a -- 1.8 a 3.5 a 
0.0 b -- -- 1.0 b 0.8 b 4.5 a 
______________________________________ 
Each line represents a balanced incomplete block experiment (4 treatments 
tested 2 at a time). All treatments contained the synergist, propyl 
acetate. The C.sub.13 tetraene (1) was used at 1.0 ng per test; the other 
tetraenes (2, 3, and 6) were used at 250 pg per test. In each row, means 
followed by the same letter were not significantly different (P &lt; 0.05, 
LSD method). 
The interactions of pheromone components are complex, and interpretation of 
wind tunnel data for certain mixtures is difficult. Nevertheless, the 
combination of 1 and 2 was always effective in the tests, and the beetles 
responded clearly to 3 and 6 in many instances. It is concluded that 1, 2, 
3, and 6 are the most biologically important male-derived pheromone 
components. 
C. lugubris 
C. lugubris beetles were field collected in oak woods and corn fields near 
Bath, Ill., by attracting them to traps each baited with individual cups 
of fermenting whole wheat dough and fermenting banana. The beetles were 
then maintained in the laboratory on standard pinto bean diet in the same 
way as C. hemipterus. Adult beetle lived as long as 6 months under these 
conditions. Volatiles were collected from C. lugubris in the same way as 
for C. hemipterus. The wind tunnel bioassay, as developed for C. 
hemipterus, worked very well for C. lugubris. With the latter species, 
however, it was not necessary to provide "host" volatiles as a pheromone 
synergist. The beetles responded very well to just the pheromone. The 
bioassays of beetle-derived preparations used approximately 5 beetle-days 
of material per test. The chromatographic, spectral, and chemical methods 
developed for C. hemipterus were also used for C. lugubris. 
As shown in Table XV, the Tenax collections from male beetles were far more 
attractive than collections from females. That the female-derived 
preparation attracted any beetles at all was probably due to volatiles 
from the beetle diet, which was present during volatile collection. 
TABLE XIV 
______________________________________ 
C. hemipterus: Comparison of Six Binary 
Tetraene Mixtures with the Standard Synthetic Mixture 
Treatment Mean Bioassay Count (n = 12) 
______________________________________ 
Control 0.8 a 
Synthetic mix 
16.4 cd 
1 + 2 21.0 d 
1 + 3 1.5 b 
1 + 6 14.2 c 
2 + 3 14.4 cd 
2 + 6 16.2 cd 
3 + 6 14.7 cd 
______________________________________ 
Balanced incomplete block experiment (8 treatments, tested 2 at a time). 
Means followed by the same letter not significantly different (P &gt; 0.05, 
LSD method). As in Table XIII, tetraene 1 tested at 1.0 ng per test; the 
others (2, 3, and 6) were tested at 250 pg per test. The synthetic mixtur 
was used at 1.3 ng per test. 
TABLE XV 
______________________________________ 
Isolation of Pheromone from Tenax Collections 
of Volatiles from Carpophilus lugubris.sup.a 
______________________________________ 
A. Comparison of Collections from Males and Females 
Source of Volatiles 
Mean Bioassay Count (n = 7) 
______________________________________ 
Male culture 14.1 
Female culture 1.4 
______________________________________ 
B. Fractionation of Tenax Collection from Males on Silica 
Mean Bioassay Count (n = 4) 
Fraction Fraction Control 
______________________________________ 
Hexane 30.3* 0.3 
5% Ether in hexane 
0.0 0.8 
10% Ether in hexane 
0.3 0.5 
50% Ether in hexane 
.sup. 2.0.sup.b 
0.5 
10% MeOH in CH.sub.2 Cl.sub. 
.sup. 3.8.sup.b 
0.0 
______________________________________ 
C. Fractionation of Hexane Silica Fraction by AgNO.sub.3 -HPLC 
Mean Bioassay Count (n = 4) 
Elution Volume (ml) 
Fraction Control 
______________________________________ 
3.0-4.5 0.0 0.3 
4.5-5.0 0.0 0.0 
5.0-5.5 0.3 0.5 
5.5-6.0 42.3* 0.0 
6.0-6.5 12.3* 0.3 
6.5-7.0 4.0* 0.0 
7.0-7.5 0.0 0.5 
______________________________________ 
.sup.a Each fraction or extract contained ca. 5 beetledays of material. N 
coattractant was added to the treatments. The symbol "*" denotes a 
statistically significant (P &lt; 0.05) difference from the control. 
.sup.b The slight activity in these fractions was due to components 
derived from the diet. 
Fractionation of the male-derived Tenax collection on silica and subsequent 
bioassays indicated that the pheromone of C. lugubris was very nonpolar 
(eluting with hexane) and was probably a hydrocarbon. 
The hexane fraction from silica was further separated by AgNO.sub.3 -HPLC. 
The fractions 5.5-6.5 ml after injection were quite active. The active 
compounds were retained on the column (column void volume was 3.0 x1), 
thus there was evidence for the presence of double bonds. In fact, active 
compounds from C. hemipterus had eluted from the AgNO.sub.3 column in much 
the same way. 
Comparison of AgNO.sub.3 fractions derived from male and female C. lugubris 
by GC revealed one male-specific peak in the active HPLC fraction. This 
corresponded in retention time to a compound encountered previously in C. 
hemipterus (retention index=15.15). The compound was identified as 
structure 6, Table I, based on mass spectrometry, hydrogenation followed 
by mass spectrometry, and comparison to four candidate synthetic compounds 
(6, 7, 11, 12, Table I). The natural compound matched structure 6 
perfectly with respect to GC retention: AGNO.sub.3 -HPLC retention; mass 
spectrum; and number, GC retentions, and mass spectra of hydrogenated 
derivatives. 
C. lugubris responded readily to 6 as well as to three of the tetraenes 
identified previously from C. hemipterus (structures 1, 2, and 3). C. 
lugubris did not respond to 5 (Table XVI); thus, C. lugubris showed much 
the same tetraene preference as C. hemipterus. 
Certain host plant volatiles sometimes synergized the effect of compound 6 
on C. lugubris. The results are shown in Table XVII. In contrast to C. 
hemipterus, aromatic esters (e.g., benzyl acetate) were effective 
individually and as synergists. Overall attractiveness could be increased 
by combining more than one host volatile. 
TABLE XVI 
______________________________________ 
Activity of Synthetic Tetraenes 
for Carpophilus lugubris 
Mean Bioassay 
Structure Count (n = 4) 
Number Tetraene Control 
______________________________________ 
1 5.8* 0.3 
2 13.8* 0.0 
3 6.5* 0.3 
5 0.3 0.3 
6 23.8* 0.0 
______________________________________ 
Each tetraene tested at 1 ng per test. No coattractant was added to the 
treatments. Significant differences between tetraenes and controls are 
denoted by (*) (P = 0.05) 
TABLE XVII 
__________________________________________________________________________ 
Synergistic Interaction Between Host Plant 
Volatiles and Pheromones of Carpophilus lugubris 
Mean Bioassay Count 
Volatile + 
Volatile Volatile 
Pheromone 
Pheromone 
__________________________________________________________________________ 
Phenylacetaldehyde 
1.1 a 10.6 b 
11.4 b 
Apple cider vinegar, ethanol, 
21.3 a 6.2 b 39.5 c 
benzyl acetate 
Benzyl acetate 3.0 a 2.2 a 8.2 b 
Ethanol, ethyl acetate, 
6.6 a 3.5 a 16.2 b 
acetaldehyde 
Methanol, water, propyl acetate, 
6.5 a 16.3 b 
29.1 b 
methyl butanoate 
__________________________________________________________________________ 
In this experiment, the pheromone was compound 6, Table I, 2 ng. Volatile 
were used at a dose of 2 mg and were formulated as 10% solutions or 
suspensions in mineral oil. In each line, means followed by the same 
letter do not differ significantly (LSD, P = 0.05) 
C. freemani 
C. freemani beetles were field collected at the same location as G. 
lugubris. The beetles were easily reared on the standard pinto bean diet 
developed for C. hemipterus. Volatiles were collected from C. freemani 
onto Tenax in the same way as for C. hemipterus. The wind-tunnel bioassay 
was used for C. freemani in the same way as for C. lugubris. Host-derived 
coattractants were not required for excellent bioassay responses; the 
pheromone alone was sufficient. 
Tenax collections from males and females of C. freemani were compared by GC 
after purification on silicic acid. As with C. hemipterus and C. lugubris, 
hydrocarbons existed that were present only in the males. The most 
abundant of these (retention index=12.2) amounted to ca. 50 ng/beetle-day. 
Another compound (retention index=15.15) was present at 3.0% of the level 
of the first compound. 
Initially, sufficient numbers of beetles were not available for highly 
replicated, quantitative bioassays; but qualitatively, the beetles 
responded clearly in the wind tunnel to the hydrocarbon fraction of the 
Tenax collection and also to AgNO.sub.3 -HPLC fractions which contained 
the most abundant male-specific hydrocarbon. The mass spectrum of this 
compound indicated a molecular weight of 164, corresponding to the 
molecular formula, C.sub.12 H.sub.20, which has three double-bond 
equivalents. Hydrogenation led to products with a molecular weight of 170. 
Thus 6 hydrogen atoms were taken up, and the original compound was 
acyclic. The derivatives with molecular weight 170 corresponded to 2 GC 
peaks. Two asymmetric centers were probably created during hydrogenation, 
and the four resulting enantiomers could produce no more than two peaks on 
an achiral GC column. The intense fragment ion (15% of base peak) at 
x/z=141 (M-29) in the spectrum of the saturated derivative suggested an 
ethyl branch. Together, the data suggested 5-ethyl-3-methylnonane as the 
carbon skeleton. By analogy to the other pheromone compounds, 
(2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene (14) was synthesized as a 
model compound for analytical comparison with the pheromone component. The 
synthetic and natural compounds were identical in every way. 
The minor component (retention index=15.15) was chromatographically and 
spectroscopically identical to the pheromone of C. lugubris and was 
therefore concluded to be compound 6. 
It was eventually possible to rear large numbers of the beetles so that 
quantitative wind tunnel bioassays could be conducted easily. As shown in 
Table XVIII, compound 14 was very active in the wind tunnel at a level of 
ca. 0.6 beetle-days (30 ng). The minor component (6) was also 
significantly active by itself, although far less active than 14, when 
tested at the same proportions as emitted by male beetles. However, when 
14 and 6 were combined, the response was over 2 times greater than for 14 
alone. Furthermore, on an equal weight basis, the combination of 14 and 6 
together was equivalent in activity to the natural, beetle-derived 
pheromone. Thus 14 and 6 together constitute the aggregation pheromone of 
C. freemani. 
Although host-derived volatiles are not required for successful wind tunnel 
bioassays, such volatiles do synergize the activity of the pheromone 
(Table XIX). Although thorough screens have not been undertaken, it is 
likely, based on the representative compounds tested, that the same 
compounds that are effective for C. hemipterus will also work for C. 
freemani. 
Applications of the Invention 
The importance of olfaction in the behavior of insects is well known. 
Insect-produced volatiles, e.g., pheromones, and host plant odors may 
facilitate location of conspecifics for mating and orientation to 
acceptable host plants for feeding and oviposition. 
TABLE XVIII 
______________________________________ 
C. freemani: Bioassay Activity 
of Synthetic Hydrocarbons 
______________________________________ 
A. Activity of compounds 14 and 6, alone and in combination. 
Treatment Mean Bioassay Count (n = 12) 
______________________________________ 
Control 0.0 d 
Compound 14 (30 ng) 
46.0 b 
Compound 6 (1 ng) 2.8 c 
Compounds 14 (30 ng) and 6 
103.8 a 
(1 ng) 
______________________________________ 
B. Comparison of beetle-derived pheromone and mixture of 
14 and 6 
Treatment Mean bioassay count (n = 8) 
______________________________________ 
Control 0.0 b 
Beetle-derived pheromone 
23.6 a 
Compounds 14 (30 ng) + 6 
26.3 a 
(1 ng) 
______________________________________ 
Three-minute tests. Balanced incomplete block experiments; in each 
experiment, means followed by the same letter not significantly different 
(LSD, 0.05). In part B, the natural ratio of compounds 14 and 6 in the 
beetlederived pheromone is 30:1, and the amounts of these compounds per 
test were the same as for the synthetic compounds. The beetlederived 
sample was from a Tenax collection and had been partially purified on 
silicic acid (elution with hexane). 
TABLE XIX 
______________________________________ 
Synergistic Interaction between Host 
Plant Volatiles and Pheromone of C. freemani 
Mean Bioassay Count 
Volatile + 
Volatile Volatile Compound 14 
Compound 14 
______________________________________ 
Propyl acetate 
0.9 a 12.6 b 38.2 c 
Ethanol 1.4 a 30.5 b 67.7 c 
Valeric acid 
0.1 a 10.4 b 21.6 c 
______________________________________ 
Compound 14 was used at 40 ng per test. Volatiles were used at 2 mg per 
test, as 10% solutions or suspensions in mineral oil. In each line, means 
followed by different letters are significantly different (LSD, P = 0.05) 
 
Pheromones that are attractive alone may have their activity enhanced or 
synergized by host plant odors which show little attraction when presented 
alone. The pheromones of this invention may be used as a crude extract of 
Carpophilus sp. beetles or in substantially purified form either isolated 
from the natural source or chemically synthesized. As a practical matter, 
it is expected that substantially pure pheromone will be formulated with 
an inert carrier for use as an insect attractant composition. 
Alternatively, the pheromone composition ray be further formulated with 
other pheromones or synergists; insecticides may also be included in the 
attractant composition to effect insect control. 
With the identification of the Carpophilus sp. beetle pheromones and 
synergists therefor, a tool is available to monitor beetle populations for 
directing insecticide applications and evaluating control measures. The 
synergized pheromones may also be potentially used to control pest 
populations by employing large numbers of traps (trap-out strategy). 
A synergist is herein defined as a material that enhances the activity of 
other materials, so that the overall activity of the mixture be is greater 
than the sum of the individual components. An effective synergist for an 
attractant pheromone facilitates insect population monitoring and control 
by increasing both the level and longevity of pheromone attractiveness. 
The compounds useful as synergists are comparatively inexpensive, and 
thereby enhance the cost effectiveness of insect control using pheromones. 
The potency of these synergized pheromone compositions dictates that they 
be applied in conjunction with a suitable inert carrier or vehicle as 
known in the art. Of particular interest are those which are agronomically 
acceptable. Alcohols, hydrocarbons, halogenated hydrocarbons, glycols, 
ketones, esters, and aqueous mixtures, and solid carriers such as clays, 
cellulose, rubber, or synthetic polymers are illustrative of suitable 
carriers. The synergized pheromone compositions may be used in a number of 
ways, e.g., in combination with pesticides to kill the insects or in traps 
to monitor population changes or to kill insects in the traps. Other 
formulations and methods of use will be obvious to skilled artisans. 
Formulation is herein defined as a physical combination of at least one 
aggregation pheromone with one or more materials selected from the group 
of other pheromones, synergistic materials, insecticides, and inert 
carriers. 
The synergized pheromone compositions encompassed herein are effective in 
attracting a variety of organisms. Without desiring to be limited thereto, 
pests of particular interest known to be susceptible to treatment are 
agronomically important insects, especially the nitidulid species C. 
hemipterus, C. lugubris, and C. freemani . 
The insect pheromones of this invention are represented by the general 
structure: 
##STR38## 
where R.sup.1 and R.sup.2 are independently selected from the group 
consisting of hydrogen and lower alkyl, and n is zero or one. Compounds 1, 
2, 3, 6, 11, and 14 (Table I) are examples of the general structure. It 
will be noted that not all examples are active for each species of 
nitidulid. It will be obvious to those skilled in the art to choose a 
compound that attracts the desired insect and an amount of the pheromone 
that will be effective. 
The arrangement of the double bonds must be in the "E" configuation, as 
illustrated in the general formula. Compounds with Z configured double 
bonds are not effective. See, for example, compounds 8 and 9 in Table I. 
It will be obvious to skilled workers in the insect pheromone field that 
the ratio and absolute amounts of active ingredients may be varied 
depending upon environmental conditions such as temperature, humidity, 
wind velocity, and insect population. 
The following examples are intended only to further illustrate the 
invention and are not intended to limit the scope of the invention which 
is defined by the claims. 
EXAMPLE 1

BIOASSAY METHOD 
All bioassays were conducted in a wind-tunnel olfactometer constructed of 
Plexiglas 0.60.times.0.60 m in cross section and 1.35 m long. The floor 
was plywood, which was rough enough in texture to allow any beetles that 
had fallen on their backs to right themselves. The ends were covered with 
30-mesh steel screen. An electric fan was connected by a duct to the 
upwind end; air was drawn from the room and forced through the wind 
tunnel. Laminar flow was achieved by passing the air through several 
layers of cheesecloth mounted outside the upwind screen, as described by 
Baker and Linn (1984). The linear air flow rate was 0.3 x/sec. The 
temperature was kept at 27.degree. ; the relative humidity was not 
controlled but was in the range of 30-40%. The wind tunnel was lighted 
from above with four 40-watt fluorescent tubes. 
About 24 hr before bioassays were to begin, cultures containing a total of 
200-400 beetles about 1-3 weeks old (except for C. lugubris which were as 
old as 6 mo) were placed in a fume hood for 8 hr, during which the diet 
medium dried down to about 75% of its original volume. The beetles were 
then transferred to the wind tunnel and kept without food for an 
additional 16 hr. The hood-drying step was omitted for C. freemani. Lights 
and air flow were left off during this time but were turned on before 
beginning bioassays. Beetles were never observed to fly to a bait unless 
they had been starved for a number of hours. For good responsiveness, the 
beetles had to have been without food but not unduly stressed. With the 
above procedure, the beetles appeared healthy and usually began to respond 
to attractive baits within 1 hr of turning on the wind-tunnel lights and 
fan. Once the beetles were ready, as many as 30-50 three-minute tests 
could be run in the course of a day. 
Test baits were suspended from a horizontal wire 0.4 m above the floor of 
the wind tunnel, perpendicular to the air flow and 0.2 m from the unwind 
screen. Baits were always tested in pairs, separated by 0.3 m. Extracts or 
chromatographic fractions to be used as baits were applied to 7-cm circles 
of filter paper which were folded into quarters and secured with a paper 
clip. Concentrations of test solutions were adjusted so that the 
application volume was in the range of 10-30 .mu.l. Because of the 
location of the baits, beetles could reach them only by flying. The test 
period was 3 min; during this time the number of beetles landing on each 
bait was recorded. Tests were always replicated and each bait was tested 
in both positions, so that any position effects would not bias comparisons 
of treatments. Tests were separated in time by 2-5 min. 
Bioassays were interpreted in terms of the mean counts of responding 
beetles. However, the counts for a particular treatment varied from day to 
day and even from hour to hour, depending on such factors as the number of 
beetles in the wind tunnel, their health, and the length of time that they 
had been starved prior to testing. To control for this variability, 
treatments are always tested two at a time in the wind tunnel. Thus, even 
if the level of responsiveness in the wind tunnel was low at the time of 
the test, both treatments showed decreased counts. Relative to each other, 
the treatments retained proper relationships. Ratios of counts between 
treatments have remained quite constant over time. Efforts were made to 
keep the level of beetle activity in the wind tunnel fairly constant from 
day to day, but comparison of mean counts is only justified within an 
experiment (not between experiments), and it is usually the ratios of 
means which ar of greatest usefulness. 
EXAMPLE 2 
EXTRACTION 
Beetles to be extracted were immobilized over ice and separated by sex 
within 7 days of emergence; then they were returned to rearing cups until 
extraction. The 30-ml plastic rearing cups normally contained up to 100 
beetles and ca. 10 ml of the pinto bean rearing medium (diet). 
As a typical example of an extraction, 300 male beetles, 9-12 days old, and 
the diet medium from the 4 rearing cups which held these were soaked in 
100 ml of methylene chloride for 15 min. The extraction was repeated twice 
more, and the combined extracts were filtered and dried over sodium 
sulfate. The extract was reduced in volume to 10 ml by rotary evaporation. 
Concentrations were calculated as beetle equivalents (the amount of 
pheromone extractable from 1 beetle) per ml, based on counts of beetles 
and extract volumes. 
EXAMPLE 3 
VOLATILE COLLECTION 
A 50-ml filtering flask was fitted with a cork into which a Tenax trap was 
inserted. The Tenax trap was prepared from a 10 cm .times.0.5 cm (ID) 
piece of soft glass tubing. A piece of brass screen (100 mesh) was sealed 
into the end by heating. The tube was filled to a depth of 0.5 cm with 
Tenax porous polymer (60/80 mesh, Alltech, Deerfield, IL) which had been 
cleaned by extraction with hexane in a Soxhlet apparatus. The Tenax was 
held in place by a plug of glass wool. About 15 ml of pinto bean diet were 
placed into the flask, and the tip of the Tenax trap was adjusted to about 
1 cm above the diet. A vacuum was applied to the Tenax trap so that 
volatiles within the flask were drawn into the trap. A second Tenax trap 
was attached to the side arm of the flask to clean the air drawn into the 
flask. This connection was made with "Teflon" tubing. Approximately 100 
male beetles were added to the flask, and the air flow through the flask 
was adjusted to 50 ml/ min. The flask was kept in an incubator at 
27.degree. and 40% relative humidity. At this humidity the diet dried out 
slowly over a week: with the diet in this condition, the beetles remained 
active and healthy, but the growth of mold was retarded. The beetles 
received 14 hr of light each day. Eighteen such flasks were operated in 
the incubator at one time. Pheromone collections were quantified in terms 
of beetle-days, defined as the average amount of pheromone collected from 
one beetle in one day. Volatile collections were also made from female 
beetles and from diet medium without beetles. 
To extract volatiles from the Tenax traps, each trap was rinsed three times 
with 200 .mu.l hexane. Before returning the trap to its flask, air was 
passed through the trap to evaporate residual solvent. Traps were rinsed 
every 2 or 3 days. The extracts were set aside for chromatography. 
EXAMPLE 4 
CHROMATOGRAPHY 
Column chromatography on silicic acid was used for all initial 
purifications. Columns were usually 5 cm by 0.5 cm, and these were 
adequate for extracts with 100 beetle equivalents, including diet medium. 
Before chromatography the methylene chloride be was carefully removed from 
these extracts under nitrogen, and the samples were taken up in hexane. 
Columns were eluted with 2 column volumes each (2 ml) with these solvents: 
hexane; 5%, 10%, and 50% ether in hexane; and 10% methanol in methylene 
chloride. Each solvent was collected as a separate fraction. Larger 
columns were used for extracts with greater numbers of equivalents. 
The rinses from the Tenax traps were also applied to these silicic acid 
columns; a collection 3000 beetle-days in size did not overload a 5 cm 
.times.0.5 cm column. 
Silicic acid containing 25% AgNO.sub.3 was also used as a packing in open 
columns (5 cm .times.0.5 cm). The samples were applied in hexane and the 
columns eluted with hexane; 5%, 10%, and 25% ether in hexane; and finally, 
with ether. 
All chromatographic separations and syntheses were monitored by gas 
chromatography (GC) using a Varian 3700 gas chromatograph. It was equipped 
with flame ionization detector, splitless injector for capillary columns, 
effluent splitter for preparative GC on a packed column, and effluent 
collector (Brownlee and Silverstein, 1968). Two columns were used: The 
first was a 15 m .times.0.25 mm (ID) DB-1 capillary with a 1.0 .mu.m film 
thickness (J & W Scientific, Folsom, CA). For many samples, this column 
was programmed from 100.degree. to 200.degree. at 10.degree. per min, 
although cooler starting temperatures or hotter final temperatures were 
sometimes required. Beetle-derived samples were usually concentrated by 
20-100 times by evaporation under N.sub.2, so that the 1-2 .mu.l 
injections would have enough material to be easily detected (&gt;1 ng per 
component). The other column, used for preparative GC, was a 2 m .times.2 
mm (ID) glass column, packed with 3% OV-101 on Chromosorb WHP 100/200 
(Alltech). The gas chromatograph was interfaced to a Hewlett-Packard 3396A 
integrator. 
Retention indices (I) relative to n-alkane standards were determined for 
the male-specific hydrocarbons. The DB-1 column was programmed from 
100.degree. to 200.degree. at 10.degree. per min, and the retention 
indices calculated by linear interpolation (Poole and Schuette, 1984, pp. 
23-25). 
High performance liquid chromatography (HPlC) was conducted isocratically 
using a Waters Associates model 6000 pump and R401 refractometer detector. 
Two columns were used. The first was a 30 cm .times.0.75 cm (ID) PLGEL 50A 
10 .mu.m size-exclusion column (Polymer laboratories, Shropshire, UK), and 
it was eluted with hexane. The other column was a 25 cm .times.0.46 cm 
(ID) Lichrosorb Si60 silica column (5 .mu.m particle size) (Alltech), 
coated with AgNO.sub.3 as described by Heath and Sonnet (1980). This 
column was eluted with 25% toluene in hexane. The void volumes for the two 
columns were estimated to be 8 and 3.5 ml, respectively. The 
beetle-derived samples were not concentrated enough to be detected by the 
refractometer. Effluent was collected as 1-ml or 0.5-ml fractions, which 
were later analyzed by GC and bioassayed. 
EXAMPLE 5 
SPECTRA 
Mass spectra were obtained on a Finnigan 4535 quadrupole mass spectrometer. 
Sample introduction was always by GC (DB-1 capillary). An ionizing 
potential of 70 eV was used for electron impact spectra. NMR proton 
spectra were obtained on a Bruker 300 mHz instrument. Samples were 
dissolved in deuterobenzene and shifts were calculated relative to 
tetramethylsilane. Further experimental details are given with results. 
Ultraviolet spectra were taken with a Perkin Elmer (Norwalk, CT) Lambda 4B 
high performance UV spectro-photometer. The solvent was hexane. 
EXAMPLE 6 
HYDROGENATION OF C-13 COMPOUND 
Saturated derivatives of male-derived hydrocarbons were prepared by the 
method of Parliment (1973), except that Methylene chloride was used as the 
solvent. Palladium (10%) on carbon was used as the catalyst in the initial 
reactions, but PtO.sub.2 was later found to be preferable because it 
caused less formation of cyclic side be products. The saturated 
derivatives were analyzed by mass spectrometry to gain structural 
information about the carbon skeletons. 
By GC, hydrogenation of the major, 13-carbon compound over Pd produced at 
least 12 distinct compounds. The key products had molecular weights of 
184; the uptake of 8 hydrogens indicated the existence of 4 double bonds 
and no rings (if no triple bonds). However, other products had molecular 
weights of 182 and would not hydrogenate further. Apparently, cyclic 
rearrangement competed with simple hydrogenation. PtO.sub.2 as catalyst 
gave a greater proportion of the acyclic product, which was more useful 
for structure elucidation. 
Mass chromatograms were prepared for the ions in the series, C.sub.n 
H.sub.2n+1 .sup.+, n=4, . . . , 12. These fragments, m/z=57, 71, 85, . . . 
, 169, were the dominant features for the acyclic products but were nearly 
absent from the cyclic products (which had C.sub.n H.sub.2n-1 .sup.+ as 
the primary series). Based on the mass chromatograms, there were four 
acyclic products (two of which were poorly resolved on the DB-1 
capillary), and these all had nearly identical mass spectra. 
The intensities of the C.sub.n H.sub.2n+1 peaks, especially those of higher 
mass, give structural information about branched alkanes (Nelson, 1978). 
These tend to fragment at branch points, with the secondary carbonium ion 
retaining the charge. Compared with the spectrum for tridecane, the peaks 
at 155, 141, 113, and 99 were relatively enhanced, while those at 127 and 
85 were relatively suppressed. These data suggested that the saturated 
derivative was 3,5,7 -trimethyldecane. 
3,5,7-Trimethyldecane possesses three asymmetric centers. If the original 
compound had double bonds involving the 3, 5, and 7 positions, then 
catalytic hydrogenation would create these asymmetric centers without 
stereoselectivity. The resulting eight optical isomers would produce at 
most 4 GC peaks on an achiral column, which is what we observed. 
EXAMPLE 7 
UV SPECTRUM OF C-13 COMPOUND 
The UV spectrum possessed a maximum at 287 nm 
(.epsilon.=2.2.times.10.sup.4) and another at 223 nm 
(.epsilon.=1.0.times.10.sup.4). The maximum at the longer wavelength 
suggested that three or four double bonds were in conjugation, but because 
steric and other factors can affect UV absorbance (Silverstein and 
Bassler, 1967), the exact number of conjugated double bonds was ambiguous. 
EXAMPLE 8 
NMR SPECTRUM OF C-13 COMPOUND 
The NMR spectrum provided important structural information, but handling 
the samples proved to be difficult. The initial NMR sample of about 20 
.mu.g was prepared by preparative GC. The purity of this sample was only 
72% by capillary GC, primarily because the target compound rearranged or 
decomposed to a significant extent on the preparative GC column. 
Nevertheless, the largest impurity was only 7% of the sample, so useful 
NMR data could be obtained. This sample was contained in a capillary NMR 
tube and was scanned 30,000 times. A subsequent NMR sample, containing 
about 30 .mu.g, was prepared by HPLC on the size-exclusion column. After 
evaporating the hexane and adding deuterobenzene, the sample was 90% pure 
by capillary GC. This sample was held in a standard (5 mm) tube, and 3200 
scans provided a good-quality spectrum. 
The spectra were difficult to interpret because the unknown compound 
rearranged, polymerized, or both during acquisition of the spectra (in the 
latter sample, totally). Peaks belonging to the original compound were 
differentiated from those due to decomposition by observing changes in the 
spectra over time. At first, no peaks were present in the region 0.8-1.4 
ppm; but over time, peaks in this area grew to become the dominant 
spectral features. Nevertheless, both NMR samples produced identical 
spectra when the artifact peaks were ignored. The observed resonances 
were: 6.25 (1H, dq, J=15.4, 2), 6.03 (2H, br s), 5.63 (lH, dq, J=15.4, 
6.7), 5.53 (lH, qq, J=6.7, 1), 2.00 (3H, br s), 1.98 (3H, br s), 1.74 (3H, 
br s), 1.73 (3H, d [half concealed], J=6.7), and 1.64 (3H, d, J=6.6). All 
the resonances appeared to represent either olefinic protons or olefinic 
methyl groups. The data suggested that the compound was 
3,5,7-trimethyl-2,4,6,8-decatetraene. The double bond at the 8 position 
had the (E) configuration because of the large coupling constant (J=15.4 
Hz) between the olefinic protons, but the configurations at the three 
trisubstituted double bonds were not determined. 
EXAMPLE 9 
(1-METHYL-2(E)-BUTENYL)TRIPHENYLPHOSPHONIUM BROMIDE (COMPOUND 24) 
In this and following synthesis examples the compounds and reagents for 
chemical synthesis were obtained from Aldrich Chemical Co. (Milwaukee, WI) 
and were used as received. Solvents were dried over 4A molecular sieves, 
except ether, which was dried over sodium metal. 
Compound 24 was prepared from triphenylphosphine (Aldrich) and 
4-bromo-2-pentene which was a previously known compound (Mulliken et al., 
1935). 
Triphenylphosphine (3.1 g, 0.012 mole) and 4-bromo-2(E)-pentene (1.7 g, 
0.011 mole) were added to 40 ml dry (molecular sieve) acetonitrile and 
refluxed for 6 hr. The solvent was removed by rotary evaporation, and the 
sticky product was washed three times with dry ether. Further traces of 
ether were removed under rotary evaporation and the product was placed in 
a vacuum desiccator for 2 hr, where it became a friable white solid. 
Alternatively, the salt crystallized after repeated (&gt;20) washings with 
dry ether, but the method using the vacuum desiccator was quicker and 
provided an acceptable reagent for the Wittig reaction. 
EXAMPLE 10 
(2E, 4E, 6E, 8E)-3,5,7-TRIMETHYL-2,4,6,8-DECATETRAENE (COMPOUND 1) 
(1-Methyl-2(E)-butenyl)triphenylphosphonium bromide from Example 9 (0.62 g, 
0.0015 mole) was added to a dry flask with 5 ml tetrahydrofuran. The flask 
was equipped with magnetic stirrer and septum; the reaction was carried 
out under nitrogen. The salt did not dissolve completely but became a 
sticky suspension. The mixture was cooled over ice, and butyllithium (2.5 
M in hexane) was added dropwise, with stirring, until the color change 
became permanent; then an additional 0.0015 mole was added. The solid in 
the flask dissolved as it was converted to the ylide. One hundred 
milligrams of (2E,4E)-2,4-dimethyl-2,4hexadienal (0.0008 mole), compound 
15, a previously known compound (Patel and Pattenden, 1985), was added to 
the Wittig reagent, and the mixture was allowed to warm to room 
temperature. The mixture was stirred for 2 hr, and it was again cooled 
over ice. Water was added dropwise until the red color of the solution had 
disappeared, and ca. 2 ml more water was added. The mixture was diluted 
with hexane and the organic layer dried over sodium sulfate. The solvent 
was removed and the product passed through a silica column with hexane. By 
capillary GC the product was 61% the (E,E,E,E) isomer, ca. 31% the 
(E,E,Z,E) isomer, and ca. 8% by-products, after clean-up on silica. 
Further purification on the AgNO.sub.3 HPLC column yielded the (E,E,E,E) 
isomer in &gt;97% purity. 
The (E,E,Z,E) isomer was recognized by its thermal lability. By GC on DB-1 
(100-200.degree. at 10.degree. /min), the (E,E,Z,E) isomer produced a 
rearrangement peak at 3.99, a sharp peak at 5.47, and a broad hump between 
these peaks. The initial peak could be eliminated by setting the injector 
temperature at 100.degree., and the hump (which indicated on-column 
thermal rearrangement) could be eliminated by using a thinner film column 
(0.25 .mu.m vs. 1.0 .mu.m), allowing the compound to elute at a cooler 
temperature (ca 115 Vs. 155.degree. ). 
EXAMPLE 11 
(1-METHYL-2(e)-PENTENYL)TRIPHENYLPHOSPHONIUM BROMIDE (COMPOUND 25) 
The compound was prepared from triphenylphosphine and 2-bromo -3(E)-hexene, 
a compound which was reported previously (Bianchini and Guillemonat, 
1968). Triphenylphosphine (1.6 g, 0.0061 mole) and 2-bromo-3(E)-hexene 
(1.0 g, 0.0061 mole) were added to 10 r1 dry acetonitrile and refluxed for 
6 hr. The solvent was removed by rotary evaporation. The thick, sticky, 
liquid product was stirred with dry ether 4 times, with the ether being 
decanted. After further traces of ether were removed by rotary 
evaporation, the protect was placed in a vacuum desiccator for 6 hr, where 
the product became a friable white solid (1.6 g, 61%). 
EXAMPLE 12 
(2E,4E,6E,8E)-3,5,7-TRIMETHYL-2,4,6,8-UNDECATETRAENE (COMPOUND 2) 
(1-Methyl-2(E)-pentenyl)triphenylphosphonium bromide (0.40 g, 0.0009 mole), 
from Example 11, was added to a flask with 2 ml dry tetrahydrofuran. The 
reaction was run under nitrogen, and the flask was equipped with a 
magnetic stirrer. The mixture was cooled over ice, and butyllithium (2.5 m 
in hexane) was added dropwise until the color change became permanent; 
then an additional 0.4 x1 (0.001 role) was added. After 5 min, 100 rg 
(0.0008 mole) of (2E,4E)-2,4-dixethyl-2,4-hexadienal (15) was added. The 
mixture was warmed to room temperature, stirred for 2 hr, then cooled over 
ice again. Water (1 ml) and pentane (3 ml) were added. The aqueous layer 
was washed twice with 2 r1 pentane. The combined organic layers were 
washed three times with water and dried over Na.sub.2 SO.sub.4. After the 
product was passed through a silica column with hexane, both the (E,E,E,E) 
and (E,E,Z,E) isomers were present (47% and ca. 40%, respectively, by GC). 
The isomers were resolved by HPLC on the silver-nitrate column, as 
described in Example 10. 
EXAMPLE 13 
FIELD RESPONSE OF C. lugubris TOWARD COMPOUND 6 AND WHOLE WHEAT DOUGH 
Traps were baited with: (1) 300 .mu.g of Compound 6 (applied to a red 
rubber septum with 300 .mu.l of xethylene chloride); (2) ca. 20 ml of 
whole wheat dough (which had been inoculated with baker's yeast); or (3) a 
combination of these two baits. Control traps were not baited. The trap 
design did not allow the captured beetles to contact the bait. The traps 
were hung 1-1.5 m above the ground in an oak woods near Bath, Illinois. 
They were placed in pairs (with ca. 1 m separation between traps), and the 
pairs of traps were separated by at least 20 m. A balanced incomplete 
block design was used. Numbers of beetles captured were recorded after 3 
days. 
The synergistic interaction between the pheromone and a food-type bait was 
more pronounced in the field (Table XX, below) than in the laboratory wind 
tunnel (Table XVII). Although the pheromone by itself was not 
significantly more attractive than the controls, the response to the 
combination of the pheromone and whole wheat dough was greater than 
20-fold the response to the wheat dough alone. The field experiment 
emphasizes the practical importance of combining pheromones with host 
volatiles for greatest effectiveness under natural conditions. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention. 
TABLE XX 
______________________________________ 
Field Response of C. lugubris 
Toward Compound 6 
Trap Bait Mean Trap Catch (n = 6) 
______________________________________ 
Control 0.0 a 
Whole wheat dough 9.1 b 
Compound 6 0.8 a 
Whole wheat dough + compound 6 
218.3 c 
______________________________________ 
Means followed by the same letter were not significantly different (LSD, 
0.05). 
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