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Team:Valencia UPV/Project/modules/methodology/EAG
From 2014.igem.org
Project > Modules > Methodology > Electroantennogram
Sesamia nonagrioides
Sesamina nonagrioides (Lepidoptera: Nocturnidae) or the corn stalk borer is an important pest of corn in Mediterranean regions and Central Africa. Feeding habitat of Sesamia larvae is the stem and the ear of a wide range of host plants, which include corn, sorghum, millet, rice, sugar cane, grasses, palms or banana [1].
These moths are nocturnal, which means that their vision is limited, so males are guided to the female from relative large distance by an odour trail opposite to the wind instead of a visual orientation. Females produce and release the sexual pheromone trough a gland present in the apex of the abdomen and the essence is transported by air currents allowing male’s receptors to detect the signal and triggering a sexual motivation response.
The S. Nonagrioides female sex pheromones blend consist of (Z)-11-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-11-hexadecenal and dodecyl acetate [2]. Nevertheless, they are not equally present in the female’s trace. The major component is the Z11-16:Ac, which presence is enough to trigger an attraction response in males, even though it is not the optimal chemical signal to draw male moths to the source.
We selected this organism to test our pheromone since the major component of the female’s pheromone blend coincides with one of the target component that The Sexy Plant is able to produce
Electroantennogram (EAG)
EAG consists of a circuit and two electrodes connected to an amplifier which is closed by means of the moth antenna. A continuous clear air flow is blown over the antenna at a constant rate and the samples to be analysed are introduced inside the air stream. Once the stimulus is exerted to the antenna, for example the pheromone, it is locked on the antenna receptors, a signal transduction cascade is initiated and it is converted to an electrical impulse, which is registered [4]. As a result, base line undergoes an up and down or derivation, similar to those shown in an electrocardiogram. Different compounds elicit different electrophysiological responses but concentration could vary the registry, too. The characterization of DNA parts involved in pheromone biosynthesis was made by transient gene expression in N. benthamiana coupled to Electroantennography (NBTE-AEG). The volatiles synthesized by the Sexy Plant were puffed into the odour delivery system and the electrical response was registred.
Figure 1. EAG Diagram
Our moths were immobilized and the antennas were cut or detached from the male. Then, terminal ends of the antenna were cut and the resulting antenna fragment was placed in the electrodes. A small amount of each sample (1 ug) was loaded onto small pieces of filter paper inside the wide section of a Pasteur pipette.
Figure 2. Antenna in contact with both electrodes
Figure 4. EAG instrument.
The characterization of DNA parts involved in pheromone biosynthesis was made by transient gene expression in N. benthamiana coupled to Electroantennography (NBTE-AEG). The volatiles synthesized by the Sexy Plant were puffed into a device containing two small electrodes connected by a moth ́s antenna. Specific receptors in the antenna respond to the pheromones producing a measurable electric output. The volatiles in our Sexy Plants induced detectable electric pulses that could indicate a pheromone response, although further testing will be required for confirmation.
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References
- Eizaguirre M, Fantinou AA (2011) Abundance of Sesamia nonagrioides (Lef.) (Lepidoptera: Noctuidae) on the Edge of the Mediterranean Basin. Psyche vol. 2012 (ID 854045) 7 pages doi:10.1155/2012/854045
- Eizaguirre M, Albajes R, López C, Sans A, Gemeno (2007) Inhibition of pheromone response in Sesamia nonagrioides by the pheromone of the sympatric corn borer, Ostrinia nubilalis. Pest Manag Sci 63:608-614.
- Howse P, Stevens I, Jones O (2004) Feromonas de insectos y su uso en el control de plagas. Davince (first edition)Traduced by Gil-Ruíz P.
- Gullan PJ, Cranston S (2014) The insects: An Outline of Enthomology. Wiley-Blackwell.
