Pheromones identification

Abstract Hymenoptera is a very large and diverse insect order that includes the majority of both the social and the parasitic insects. With such diversity comes a variety and complexity of semiochemicals that reflect the varied biology of members of this order. This chapter reviews the chemical identification of pheromones and semiochemicals in the order Hymenoptera since 1990. For this review, the species in Hymenoptera have been classified as solitary, parasitic, or social. The chemical diversity of semiochemicals in Hymenoptera and future trends in pheromone identification are also discussed. 

This chapter reviews the literature of semiochemical (mostly pheromone) identification in Hymenoptera published since 1990. For this review, we separate the order Hymenoptera into the following three, somewhat overlapping, classes to reflect their differences in biology and semiochemistry solitary, parasitic, and social (Table 1). Although there is considerable literature on the semiochemical activity of specific glandular extracts and the chemical composition of specific glands, only those chemicals with demonstrated pheromonal (or semiochemical) activity will be specifically discussed here. The earlier literature of pheromones in social hymenoptera has previously been reviewed [4-6]. There have been more recent reviews of pheromones in social hymenoptera [7-10], parasitic wasps [11,12], sawflies and seed wasps [13,14], and mating pheromones across Hymenoptera [15]. 

Pheromone identification is still difficult because the structure of unique compounds present in small amounts in mixtures of similar molecules has to be elucidated. This topic will be discussed in detail by Ando as well as by others, showing nicely the recent progress in analytical techniques. The following chapter by R. Jurenka deals with insect pheromone biosynthesis with special emphasis on lepidopteran pheromones and also covers genetic aspects. The subsequent chapter by C. Keeling et al. describes the hymenopteran semio-chemicals (bees and ants), describing pheromones and allelochemicals. The hymenoptera add a certain flavor to the scene, because now the complexity of social insects with their many interactions comes into play, as well as the multi-level (multi-trophic) signals used by parasitoids. 

In the sections that follow, the various bug families are dealt with in their commonly accepted taxonomic order [3]. It should be noted that pheromones have been described from less than half of the bug families. Even for those families in which pheromones are known, pheromone identifications have been carried out for only a few species, leaving a vast number of semiochemicals still to be discovered. 

Female volatile pheromone (-)-periplanone-B The identification of the sex pheromone of P. americana has not been straightforward. A brief historical account provides a good case study of the possible problems associated with pheromone identification (Stinson, 1979 Schal and Smith, 1990). Roth and Willis (1952) discovered that females release an odorant, minute amounts of which stimulate male courtship. This pheromone could be extracted with ether from filter paper taken from containers that housed virgin females. Wharton et al. 

Detecting redundancy in a chemical communication system requires more rigorous testing of blends than is common in pheromone identifications. Typically, such identifications focus on the minimum number of compounds to duplicate the behavioral response stimulated by a female moth. A subtraction bioassay that documented no adverse effect of deletion of a compound could lead to the conclusion that the compound is not a pheromone component. Clearly redundant components could be overlooked in this way. Perhaps for this reason, very few cases of redundancy have been documented (but see Linn et al., 1984 Rhainds et al., 1994 King etal, 1995). 

Almost a decade before the term pheromone was coined, Roth and Willis (1952) conducted seminal experiments that characterized volatile and contact pheromones in cockroaches. Louis Roth s research integrated studies of endocrinology and behavior, and the influence of this approach was reflected in Barth s early articulation of the interplay between the endocrine system and sexual behavior. In later years cockroaches continued to serve as important models for invertebrate endocrinology (Scharrer, 1987 Tobe and Stay, 1985), but research on pheromones lagged, in part due to technical difficulties in sex pheromone identification. Below, we highlight some of the many issues yet to be resolved in the physiological and behavioral regulation of sex pheromone production and emission in cockroaches. 

Smith, R.G., Daterman, G.E. andDaites, G.D. (1975). Douglas-fir tussock moth Sex pheromone identification and synthesis. Science, 188, 63-64. 

Microchemical reactions, which with care and suitably sized microscale equipment can be carried out on nanogram amounts of material, can be used to determine the presence or absence of specific functional groups, or determine the numbers, positions, and even geometries of double bonds. The application of microchemical reactions to pheromone identification has been reviewed in detail by Attygalle (1998). Coupled GC-Fourier transform infrared spectroscopy has also found occasional use in pheromone identification (Attygalle et al., 1995 review, Leal, 1998). 

Grant, G. G., Millar, J. G., and Trudel, R. (2008). Pheromone identification of Dioryctria abietivorella (Lepidoptera Pyrallidae) from an eastern North American population geographic variation in pheromone response. Can. Entomol. 141 129-135. 

In either case a pheromone identification should be followed by an investigation of the response of other species to synthetic compounds, blends, isomers, enantiomers, etc., and comparison of the results with the natural interspecific responses or behavior. Additionally, odors from host plants or pheromones of other species may affect the response of members of a species to their pheromones. 

In at least some cases where single component pheromones have been identified the pheromone identification probably can be labeled incomplete. As more research is conducted on the behavior of these species and their interactions with other species, particularly those in the same family and genus, more compounds will undoubtedly be identified that are active in mediating the behavior of these species. At this time I can only reiterate that considerably more research is needed on coleopterous pheromones before we can begin to understand these complex interactive systems. For natural products chemists this should be a fruitful area for investigation for some time. 

A key priority for the future is the continued collaboration between crustacean researchers and chemists - for both pheromone identification and synthesis. There are good signs of this happening. The next decades may be among the most exciting yet. 

It seems evident that to achieve pheromone identification, a clear biological assay relying upon unambiguous behavior coupled with a chemical identification strategy, testing each successive purification step, is critical. Additionally, the use of biologically relevant samples such as conditioned seawater that contains compounds released into the environment at biologically relevant concentrations should be used. 

Carcinus maenas, an Ideal Organism to Attempt Pheromone Identification... 

For anyone attempting pheromone identification, this diverse range of techniques and potential pitfalls represents a difficult and complex challenge to select the appropriate purification, fractionation, and identification strategies relevant to the compounds under investigation. 

Molecular identification of sex pheromones in marine crustaceans has proven to be very difficult, to the point that no unequivocal identification for any decapod crustacean has been published (but see Hardege and Terschak, Chap. 19). Several factors have made successful identification difficult. Some difficulties are common to searches in other animals pheromones are likely blends of molecules whose components are at very low concentration. In addition, pheromones of marine crustaceans are often small and polar molecules, making them difficult to separate from salts and other small ions in their background, i.e., sea water or urine. These difficulties have led us to take on new approaches to pheromone identification and to select the blue crab C. sapidus as our experimental model. 

Muller-Schwarze, D., Muller-Schwarze, C., Singer, A. G., and Silverstein, R.M. 1974. Mammalian pheromone Identification of active component in the subauricular scent of the male pronghorn. Science 183, 860-862. 

Insects have been ideal organisms on which to carry out semiochemical studies and as a majority of their pheromones are volatile or semivolatile they are particularly suited to GC-MS analysis. This, coupled with the slower introduction of LC-MS explains why the use of HPLC in pheromone identification is more limited however, this is changing. A recent report used the greater resolving power of chiral LC compared to chiral GC to directly measure the natural ratio of enantiomers of (Z,Z)-cis-3,4-epoxy-6,9-non-adiene, the female sex pheromone of the Japanese giant looper moth. This article also suggests that with the use of microcolumns LC-MS has the potential to rival GC-MS for sensitivity, thus taking HPLC out of its previous use of prefraction prior to GC-MS analysis and the purification and separation of synthetic pheromones. 

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