Hence the idea of working on the click beetle's larval stage: the wireworm. And no small task this is! ‘The expertise we have in Gembloux on airborne odours is well known in Belgium, and perhaps even in Europe. It has only been a few years, starting with the Rhizovol project (identification and quantification of volatile organic compounds released by barley roots when attacked by pathogens), since we've began looking into the field of underground odours, which is much more complex! While airborne odours can be carried by the wind over several kilometres and contribute, for instance, to the sexual attraction of insects, this is not the case underground, where odours are carried over a few dozen centimetres only. A myriad of obstacles can hinder their diffusion, starting with water, dirt and other particles: this makes integrated pest management more complex. In addition, a methodological challenge presents itself as early as the experimental stage: how to see the reactions of underground insects when they are exposed to a certain stimulus? While chemists and entomologists have long been aware that the CO² released by the roots alerts insects that there is animal or plant life nearby, this signal remains a very generic one that says nothing about whether a given plant attracts or repels pests. In the specific field of wireworms, no progress had been made in many years, at least not as far as odours are concerned.’ 

Soil: a new challenge for researchers

This situation has been evolving, starting with the work of Fanny Barsics, a former doctor at the entomology unit - whom successfully defended her thesis in February 2015. Under the supervision of François Verheggen, this young researcher has managed to isolate which odours released by spring barley roots (Hordeum distichon), one of the most targeted species of plant, attract wireworms (Agriotes sordidus). ‘Odour’ refers here to a combination of organic molecules with low molar mass that are volatile at room temperature and can activate the olfactory system of a receiving organism. These can also be referred to as ‘semiochemicals’, i.e. molecules that can transmit information to a receiver. ‘The challenge is to use well-known methods for capturing airborne odours, and adapt them to an underground environment,’ explains François Verheggen. ‘Soil is a complex environment that includes matter in the solid, liquid and gaseous states. Any volatile molecule can leave the air contained in the soil, dissolve in the water that infiltrates it, or attach itself to solid particles. In order to overcome this methodological challenge, we have received valuable help from Marie-Laure Fauconnier, head of Gembloux Agro-Bio Tech's General and Organic Chemistry department, and her doctoral student Benjamin M. Delory. Based on the “dynamic head-space sampling” approach, the method they used to capture odours was presented in an article published in Plant Physiology and Biochemistry in 2016(2). In conjunction with gas chromatography, using barley root samples and crushings, this method enabled us to identify four molecules – more specifically four aldehydes (organic molecules containing one carbon/oxygen/hydrogen group) – released by the roots: hexanal, (E)-hex-2-enal, (E)-non-2-enal, and (E,Z)-nona-2,6-dienal.’

Once this step was completed, the researchers demonstrated that these four allelochemicals (i.e. semiochemicals involved in an interaction between two different species) are among the substances that can act upon the wireworm's sensory system and guide them to the barley’s roots. As they could not rely on traditional olfactometres, which are designed to capture airborne odours, the two researchers designed specific olfactometres to observe the behaviour of underground larvae. They are built in a Y-shaped structure, and consist in a 30-centimetre-long tube with an opening in the middle where the wireworm can be inserted. At one end, the researchers inserted a substrate where a barley seed could germinate and develop. The other end remains open. Then, at one end of a second set of olfactometres, the researchers inserted an odorous mixture that included the four aldehydes diluted in triacetin in the same proportions as identified during the first stage of the research: 47% (E)-non-2-enal, 31% (E,Z)-nona-2,6-dienal, 11% (E)-hex-2-enal, and also 11% hexanal. In terms of dosage, the wireworms were exposed to three different odorous baits: 10 micrograms, 1 milligram, and 100 milligrams. The wireworms' movements were made easier by coating the olfactometres used in the experiments with vermiculite, a brittle, chemically inert, and low-density insulating material. 

(2) Barley (Hordeum distichon L.) roots synthesise volatile aldehydes with a strong age-dependent pattern and release (E)-non-2-enal and (E,Z)-nona-2,6-dienal after mechanical injury, Benjamin M. Delory, Pierre Delaplace, Patrick du Jardin, Marie-Laure Fauconnier, Plant and Soil, 2016.