Department of Chemistry, Cardiff University, P. O. Box 912, Cardiff, CF10 3TB, Wales, UK; KellyDR@Cardiff.ac.uk

If we take the date of publication of the structure of bombykol [1] as the birth of semiochemistry, then today, it is reaching middle age. However this does not indicate that the area is mature or slowing down, indeed the pace of developments continues to accelerate, with advances from a host of new techniques and approaches [2]. This morning's session intentionally reflects many disparate aspects of semiochemistry, including agricultural applications, structural elucidation and molecular biology. On one hand, the determination of the structure of semiochemicals can now be achieved in a reasonable time (< one research grant), given an adequate supply of materials, a suitable bioassay and some luck! However the role of a given semiochemical and its relationship to other semiochemicals, plus visual and auditory cues and behavioural imperatives is more difficult to determine. This is shown most clearly in the use of pheromones for pest insect control, on agricultural crops, which has not fulfilled its early promise. In the first lecture, John Pickett describes recent developments in push-pull strategies for diverting pests from crops and the science which underpins it [3]. This approach uses low inputs and hence can form a component of a sustainable agricultural system. On the other hand pheromones have been effective for monitoring insect pests and mating disruption of moths. The successful deployment of a pheromone based monitoring or control system requires careful exploitation of the factors described above, and all at an economically viable price! These issues are discussed in the lecture by Owen Jones, the founder and chairman of Agrisense BCS [4]. The last four lectures are a pot pourii of topics from the cutting edge of semiochemistry.

The sex pheromones of the social Hymenoptera have been investigated in exquisite detail, but the non-social species have been neglected, although many attack the seeds of economically important crops. Basilios Mazomenos has turned his attention from olive pests [5] to the almond seed wasp, Eurytoma amygdali and has discovered a potent male attractant.

The black truffle has delighted gourmets for centuries and has been the source of a considerable body of folklore. The ability of dogs, pigs or insects to locate truffles has also provoked much speculation. Following on earlier work with fungus volatiles [6], Thierry Talou has started to give the folklore a scientific explanation. In a study which encompasses plant, insect and mammalian semiochemistry, the key attractant for truffle hunting animals has been identified.

Some years ago, I proposed the first law of semiochemistry. Which states that "our knowledge of the semiochemistry of a species, is inversely proportional to its size"! The examples of this law are legion: we know more about the semiochemistry of insects, than all other organisms, put together. On the microscale, yeast pheromones are well understood, whereas mammalian semiochemistry is dominated by examples from mice and rats. Very little is clearly understood about larger mammals, including important domesticated animals such as cats, dogs, cows and horses, although the pig pheromone has been used commercially for several years. Thus far, there is only one exception to the first law: the Asian elephant oestrus signal [2], however I am fairly confident that the pheromones of the blue whale, Balaenoptera musculus, may not be discovered for some years. More seriously, the semiochemistry of mammals has only really begun to make significant progress in the past 10-15 years and Raimund Apfelbach has made pioneering contributions [7]. In the lecture he will describe a unique study, made with Russian collaborators. The dwarf hamsters Phodopus campbelli and P. Sungorus have sacs at the openings of the cheek pouches, which are used to mark food stored in the pouches. The secretion from these sacs is essential for the development of the young and acts as a marker on food stores. Thus these secretions provide an excellent opportunity to investigate pheromonal materials which are essential for survival of the species. The essential role of odorant binding proteins (OBPs) in mammalian (and insect [8]) semiochemistry has emerged over the past 10 years, as a consequence of the extraordinary developments in molecular biology. It is becoming clear that pheromonal OBPs, signal their presence by release of a volatile ligand (the pheromone's pheromone [2]), which attracts a conspecific and promotes sniffing and licking. This causes transfer of the pheromonal OBP to the target organ, which is usually the vomeronasal organ. Aphrodisin is an OBP of the lipocalin family of proteins, which facilitates copulatory behaviour by the male golden hamster, Mesocricetus auratus. Jean-Claude Pernollet has achieved heterologous expression and purification of aphrodisin, which should enable "large-scale" production and a thorough investigation of its properties.

J. A. Pickett and L. J. Wadhams

Biological and Ecological Chemistry Department, IACR-Rothamsted, Harpenden, Herts., AL5 2JQ, U.K.

Contrary to widely extant views, olfactory interactions relating to the location of plant and animal hosts are based principally on highly specific olfactory neurons as opposed to generalist molecular receptor systems. This has underpinned the demonstration that host location relies on the detection of specific compounds representative of suitability in host taxa or physiological state, and more recently that morphological features at the peripheral olfactory neural system may allow accurate determination of the relative composition of semiochemical mixtures. Furthermore, it is now evidenced from examples of different insect Orders that the avoidance of unsuitable hosts, again either by taxa or physiological state, also involves recognition of specific molecular structures associated with specific olfactory neurons. In addition to taxonomic differences that determine host suitability, the issue of stress causing induction of novel semiochemicals or increased release is now seen to play a prominent role. With this understanding and the ensuing identification of host and non-host semiochemicals has come the greater ability to exploit semiochemically-mediated interactions in crop protection. Thus, push-pull or stimulo-deterrent diversionary strategies can be employed in which colonisation of plant or animal hosts is reduced by deployment of non-host semiochemicals, while at the same time pests are aggregated onto trap plants or animals where attractant semiochemical release is maximised. Such push-pull strategies can be managed by use of slow-release semiochemical formulations and considerable success has recently been achieved in subsistence agriculture by use of living plants to produce, directly, the push-pull effects in order to control immigration of insect pests. Although the latter is suitable for low-input sustainable agricultural systems, only by understanding the underpinning neurophysiology and associated chemistry can the approach be made sufficiently robust and sustainable for widespread development.

AgriSense BCS Ltd, Treforest Industrial Estate, Pontypridd, Mid Glamorgan, CF37 5SU, U.K.

The Semiochemical Industry today has total world-wide sales at the producer level of about U.S.$60 - 70 million. The majority of this business is split equally between North America (US, Canada and Mexico), ENAME (Europe, N. Africa and Middle East), and the Far East (Japan, India and China predominantly). Over 60 % of the Semiochemical market is made up of the sales of about a dozen companies, over half of which are based in North America. It has taken nearly 25 years for this market to grow to its current level but it still constitutes no more than 1% of the world insecticide market ($7 billion). The sale of traps and lures for monitoring insect pests accounts for nearly a half of the Semiochemical Market, while the bulk of the remaining sales come from mating disruption products for moth pests.

Whereas the expectations for the insect Monitoring market were never very great, the market for Control products based on Semiochemicals was always projected to be great, but very difficult to realise in practice. Many start-up companies have entered the market historically and a number have failed, withdrawn from the market or been sold to other bio-pesticide companies. Those that are active in this market today have had to develop strategies which give the best chance of success given the limitations on the technology while at the same time allowing for the relatively slow pace of development of the market.

Three factors have been paramount in determining the rate of development of the insect control market using semiochemicals:

(1) The reliability and robustness of the technology, (2) Its cost effectiveness and (3) the regulatory requirements for semiochemical-based product registrations.

The industry’s understanding of the technology has improved greatly over the last ten years so that the technology’s limitations can now be taken into account when applying the products in the field. Area-wide programmes have been the key to the success of the technology in crops such as cotton and apples. Much has been done to improve the synthetic processes for manufacturing the semiochemical active ingredients and their formulation for controlled release has moved on greatly. The regulatory authorities in most countries have also taken an enlightened view when it comes to the registration of products based on pheromones; this has helped greatly in mitigating the registration cost element of what is essentially a species-specific product.

This paper reviews the current trends in both the ‘monitoring’ and the ‘control’ market for pheromones and other semiochemicals and looks at possible future developments which may further influence the growth of this market.

Basilios E. Mazomenos and Fragoulis D. Krokos

Chemical Ecology and Natural Products Lab. Institute of Biology NCSR "Demokritos" P.O. Box 60228 Ag. Paraskevi Attikis, Greece 153 10, e-mail: bmazom@mail.demokritos.gr.

Introduction: Research on the chemical communication, in Hymenoptera, has mainly focused on the social insects and little information is available for other families. The family Eurytomidae (Hymenoptera) includes many parasitoids and some herbivorous species, which attack the seeds of very economically important crops e.g. almonds, pistachio, prickly custard apple, alfafa. The herbivores present much interest regarding the chemical communication system employed, since many species reproduce asexually whilst others reproduce sexually. Little knowledge on the chemical communication of these herbivore species is available. Oct-1-en-3-ol, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, (Z)-3-hexen-1-yl acetate, (E)-β-farnesene and caryophyllene released from the host plants affect host finding and ovipositional behaviour of the two sibling alfalfa and clover seed wasps Bruchophagus roddi and B. gibbus¹. Plant volatiles also affect the oviposition preference of the almond seed wasp Eurytoma amygdali². Evidence for chemicals cues used for their sexual communication so far was reported for the species Bephratelloides pomorum³ and Eurytoma amygdali⁴, however the sex pheromone compounds were not identified. We report the identification of the sex pheromone compounds of the Eurytoma amygdali. The active compounds were isolated from the crude extract by preparative fractionation of the crude hydrocarbons on a silver nitrate impregnated silica gel column, which efficiently separates: alkanes, alkenes and alkadienes. The highest male response was elicited by alkadienes and the lowest by alkenes with the alkane fraction being inactive. The identification of alkenes and alkadienes was based on gas chromatographic, mass spectrometric and gas-phase infrared data. Bioassays suggest that the two alkadienes, (Z,Z)-6,9-C_23:2 and (Z,Z)-6,9-C_25:2 and to a lesser extent alkenes produced by females are potent male attractants.

[1] Light, D. M., Kamm, J. A. and Buttery, R. G. (1992). J. Chem. Ecol., 18, 333-352.

[2] Koulousis, N. A. and Katsoyannos, B. I. (1994). Ent. Exp et Appl., 73, 211- 220.

[3] Leal, W. S., Moura, J. I. L., Bento, J. M. S., Vilela, E. F., Pereira, P. B. (1997). J. Chem. Ecol., 23, 1281-1289.

[4] Katsoyannos, B. I., Koulousis, N. A. and Bassiliou, A. (1992). Ent. Exp. et Appl., 62, 9-16.

T. Talou, C. Raynaud and A. Gaset, INPT-ENSCT, 118 route de Narbonne F-31077 Toulouse email: talou@cict.fr mailto:talou@cict.fr

Introduction: Black Truffles are the fruiting bodies of the hypogeous fungus Tuber melanosporum Vitt. which grow in symbiosis with certain trees, especially oaks. They are harvested with pigs or specially trained dogs, or by observing a particular fly (genus Suillia) in several parts of Italy, Spain and France. The typical flavor of fresh black truffle is very much appreciated by gourmets to the extent of being called "The Black Diamond" of French Cuisine. Despite its economic interest, at the beginning of the last decade, few works had been performed on black truffle aroma volatiles analysis especially for key-flavor compounds identification. In the same idea, the aromatic chemicals responsible for underground truffle localization by truffle hunting animals were unknown. The present paper reports a global approach for identification of fresh black truffle odor key-compounds by combining instrumental, behaviour and electrophysiology analysis among human experts from truffle industry and truffle hunting animals (pigs, trained truffle dogs and Suillia species flies).

Experimental: Chemical identification of flavor key-compounds was performed by using Gas Chromatography combined with Dynamic HeadSpace Concentration and coupled with Mass Spectrometry and Olfactometry. Analysis were carried out on freshly harvested truffles and the odorous effluent from GC column was assessed by experts according to the olfactory referential "The Field of Odours". Different samples of oily based mixtures of chemicals identified as the major black truffle volatiles, and Nature-Identical Truffle flavouring were buried in truffle field soil for the behaviour study among sows, boars and piglets, trained dogs and 3 Suillia species flies. Complementary tests with genuine truffle, 5 -androstenol (steroïd identified in truffles) and various volatiles (odorous but not identified in truffles) were performed. Electrophysiology study is based on electroantennographic recording of sexually mature flies heads (males and females) in presence of odorized air stream from diluted parafin solutions of the single identified truffle volatiles.

Results and discussion: More than 50 volatiles components were identified in black truffles [1] for which less than nine are reported by human experts to be key-flavor compounds [3]. Dimethyl sulfide appeared to be the key-odor compound for truffle localization by truffle hunting animals [2] but two other sulfurous, and three C₈ compounds were reported to be attractive for truffles flies [3].

References:

[1] Talou, T., Delmas, M. and Gaset, A. (1989), in "Flavors and off Flavors’ 99", (Ed. Charalambous, G.), pp1308-1315.

[2] Talou, T., Gaset, A., Delmas, M., Kulifaj, M. and Montant, C. (1990), Mycological Research, 94, 277-278.

[3] Talou, T. (1992), Doctoral Thesis INPT.

R. Apfelbach¹, U. Schmidt¹ and N.Y. Vasilieva²

1Universität Tübingen, Institut für Zoologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany; e-mail: Raimund.Apfelbach@uni-tuebingen.de.