Laboratory pheromone bioassays

The pheromone chemistry of A. hilare has been studied in detail, using both laboratory (vertical Y-tube) and field bioassays [75]. In Y-tube bioassays, mature females were attracted by male odors, but males were not attractive to other males, and females were not attractive to either sex. Males produced (-)-cis-Z-BAE and (-)-frazzs-Z-BAE in a -19 1 ratio, and other compounds in the extracts were not active. The 19 1 blend was more attractive to females than other ratios, and the individual components were not attractive, indicating that both compounds were required. Females were attracted to the synthetic blend in field cage trials [75],but as with most other phytophagous pentatomids,few bugs were caught in pheromone-baited traps (J.G. Millar and H.M. McBrien, unpublished data). 

Almost simultaneously with the identification of 2E, 4 , 6Z- 10 COOMe, a thermally unstable stereoisomer, 2E, 4Z, 6Z- 10 COOMe 118, was found to be a key component of the male-produced sex pheromone of Thyanta pallidovirens> along with the sesquiterpenes (+)-a-curcumene, (-)-P-sesquiphellandrene, and (-)-zingiberene 119 [10,25]. 2E, 4Z, 6Z-10 COOMe was an essential component of the attractive blend, whereas any one, any two, or all three of the sesquiterpene components were equally effective as the other portion of the blend. None of the components were active alone. Pheromone blends attracted only females in both laboratory and field bioassays [10]. The same compounds are also produced by the congener T. custator [10] and other Thyanta spp. (J.G. Millar, unpublished data). 

The synthesis of pheromones remains an important cornerstone of pheromone research and development. The initial synthesis of an insect pheromone serves not only to confirm the structural identification, but also provides authentic material for either laboratory or field bioassay. Oftentimes, larger quantities of the pheromones are desired for the development of monitoring and pest control methods. The scale up of the synthesis of complex pheromone compounds is often a major bottleneck in the practical application of pheromones. Also, the unusual structure of some pheromone compounds beckons synthetic chemists to apply new methodology in order to show off their methods. 

The male produced sex pheromone of the red-shouldered stink bug, Thyantapallidovirens Stal (Hemiptera Pentatomidae), was shown to consist of a blend of methyl ( "2, Z 4,. Z6)-decatrienoate and the sesquiterpenes (+)-a-curcumene, (—)-zingiberene and (—)-P-sesquiphellandrene. In laboratory bioassays, sexually mature males attracted sexually mature females but not males, and females did not attract either sex. Pheromone... 

Smith, R.G. (1976). Laboratory and field bioassay of the Douglas-fir tussock moth pheromone, (Z)-6-heneicosen-ll-one. Environ. Entomol., 5,1187-1190. 

The Z isomers were generally not active in the wind-tunnel bioassays (Bartelt et al., 1990a, 1992b), but large amounts of the synthetic (2E,4E,6Z) isomer of triene 2, which was never found in natural samples, did partially inhibit the response of Ca. freemani to its major pheromone component (all-E 2) in the wind tunnel (Petroski and Weisleder, 1997). Petroski and Weisleder (1999) showed that certain unnatural, cyclopropyl or meth-oxy analogs of triene 2 could greatly reduce laboratory responses of Ca. freemani to its pheromone. 

It has been discovered that the crucifer flea beetle, Phyllotreta cruciferae Goeze, a significant pest of oilseed Brassica and other cruciferous crops in North America and Europe, uses a hydrocarbon pheromone. The first published evidence for a pheromone in P. cruciferae was that canola plants infested by unsexed adults were more attractive to both males and females than damaged plants only, in both laboratory and field bioassays (Peng and Weiss, 1992). Subsequently, Peng et al. (1999) determined with field bioassays that the males were the attractive sex, fitting the pattern of a male-produced aggregation pheromone. 

There are other examples of volatile hydrocarbon pheromones in beetles that have been researched less extensively. One of these is from the broad-homed flour beetle, Gnatocerus cornutus (F.) (Coleoptera Tenebrionidae), a common stored-product pest. Tebayashi et al. (1998) found that males produce at least one compound that was attractive to both sexes in laboratory bioassays and at sub-nanogram levels. The compound was identified as the sesquiterpene (+)-acoradiene. The beetle-derived enantiomer was subsequently synthesized... 

However, whereas the silkworm female appears to attract males with a single sex pheromone, many other insects use blends of pheromones as chemical releasers of behavior. This phenomenon is strikingly illustrated in the case of males of the bark beetle Ips paraconfusus (=Ips confusus) which utilize three monoterpene alcohols as an aggregation pheromone (11). Maximum attraction of beetles in the field was exhibited in the presence of a mixture of all three compounds, whereas single or pairs of compounds were considerably less active (12). Similarly, in laboratory bioassays, mixtures of compounds were vastly superior to single constituents as attractants (13). 

Reliable bioassays are indispensible for a successful isolation and identification of semiochemicals, especially pheromones. Recent improvements range from the construction of new olfactometers102 and the development of a delivery system for laboratory bioassays103 to measurements of odor plume structures in the wind tunnel104 and to a new method to improve olfactory responses to gas... 

The bioassay of (l/ ,55,7R)-(+)-exo-brevicomin (76) and its (-)-enantiomer 16 ) was carried out by Professor David L. Wood and coworkers at the University of California, Berkeley. When I visited his laboratory in June 1974, I was deeply impressed by the fact that only (+)-76 was pheromonally active to attract the beetles. A large-scale field test also indicated that (+)-76 was the bioactive isomer, while (—)-76 was neither active nor inhibitory.32 Thus, only a single enantiomer of the pheromone was found to... 

Four species of Trogoderma beetles use methyl-branched alkenals as their major sex pheromones 300). Aeration of T. gramrium and trapping of the volatiles on Porapak Q gave a 92 8 ratio of (Z)- to ( )-14-methyl-8-hexadecenal. The Z-isomer was the major component obtained from T. inclusum and T. variabile and the -isomer was obtained from T. glabrum. In laboratory bioassays males could discriminate between the geometric isomers. 

Gnathotrichus sulcatus produces 6-methyl-5-hepten-2-ol (sulcatol) (229) as its aggregating pheromone, as a 65 35 mixture of 5-(+) and R- —) enantiomers (31S). Borden et al. (316) found that this ambrosia beetle responded maximally to a racemic mixture (50 50) of the two enantiomers. In fact, the response to the racemic mixture was significantly greater than it was to a 65 35 ratio. More recent work, however, suggests that G. sulcatus responds to some extent to 5-(+)-sulcatol alone, that it does not respond to the 5 -(—)-isomer, and that the range of active ratios is much wider than previously indicated (317). In addition, Borden and coworkers (317) have found that G. retusus responds to (5)-(+)-sulcatol in an upwind laboratory bioassay and that the response appears to be inhibited to some extent by the S-(—)-enantiomer. They speculate that speciation may depend in part on the enantiomeric compositions of the pheromone. 

The response of the European elm bark beetle to isomers of its pheromone blend was determined with two separate laboratory bioassays and field tests (93, 318). A combination of 3 compounds that act syner-gistically was found (-)-4-methyl-3-heptanol (294a), (-)-a-multistriatin (303a), and (—)-a-cubebene (283) (for formulas, see pp. 48, 49) (93). The absolute configuration of the (—)-a-multistriatin is IS,2R,4S,5R (49) and that of the (—)-4-methyl-3-heptanolis 38,48(147). Both the texam alarm pheromone, (5)-(+)-methyl-3-heptanone, and the alcohol above share 45 stereochemistry. Of interest is the fact that the reduction of decalones by microbial enzymes is usually stereoselective, with optically active alcohols of 5-configurations being obtained (319). 

Birch and coworkers (327) have shown in laboratory bioassays that female Ips pint from the western USA respond well to (—)-ipsdienol (266a, Scheme 52) but do not respond to either the (-l-)-isomer (266b) or a racemic mixture. This contrasts with the eastern I. pini which responds to the (-i-)-isomer and a racemic mixture but does not respond to (-)-ipsdienol (328). The mechanism for the intraspecific variation in pheromone systems of I. pini described by Lanier et al. (329) seems partially resolved. Plummer et al. (45) found that the natural enantiomer composition of ipsdienol for I. pini from Idaho to be 100% (—). The European fir engraver, Pityokteines curvidens, aggregates only in response to the 5 -(-)-isomer of ipsenol, and the i -(-l-)-isomer is inactive (330). 

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