Team:Valencia UPV/Project/modules/methodology/EAG

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<p><h3 class="hook" align="left"><a>Project</a> > <a>Project Modules</a></h3></p><br/>
 
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Antenograma
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<p><h3 class="hook" align="left"><a>Project</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology">Methodology</a> > <a>Electroantennogram</a></h3></p><br/></br>
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<div align="center"><span class="coda"><roja>S</roja>ample <roja>P</roja>reparation: <roja>H</roja>eadspace <roja>S</roja>PME</span> </div><br/><br/>
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<p class="subpart">Sesamia nonagrioides</p>
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<p>
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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]. </p>
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<p>
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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. </p>
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<p>   
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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. </p>
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<p>
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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
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</p><br/><br/>
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<p class="subpart">Electroantennogram (EAG)</p>
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<pEAG is a detection system that records the potential difference that arises from an exposure to a chemical signal. It detects and measures the quantitative and qualitative response of insect antennal receptor cells to a particular biologically active compound employing a moth antenna. The antenna is the site of olfaction of the moth, composed by antennal receptors which are very sensitive and specific so they enable perception and recognition of particular odours. In this case a male’s moth antenna is used because male moths are capable of receive and process the essence that female emit into the environment. The pheromone stimulus exerts a determined electrical response which is registered and quantified in volts. </p><br/><br/><br/>
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<p>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 <i>N. benthamiana</i> 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.</p><br/><br/>
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<div align="center"><img width="550px" src="https://static.igem.org/mediawiki/2014/a/a7/VUPVSample_prep1.png" alt="solid_phase_extraction" title="Solid Phase Microextraction"></img></div><br/>
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<div align="center"><p style="text-align: justify; font-size: 0.8em; width: 670px;"><b>Figure 1</b>. Schematic representation of the headspace solid-phase microextraction adsorption/desorption. In the adsorption step the fiber is exposed in the headspace of a vial containing biological material, where volatile organic compounds (shown in red) are adsorbed by the fiber. In the desorption step, the fiber is introduced in the Gas Chromatograph injection port where the volatile compounds are released by thermal desorption.</p></div><br/>
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<p>We have used HS-SPME coupled to Gas Chromatography-Mass Spectrometry detection to analyse the presence of pheromones (<span class="red-bold">(Z)-11-hexadecen-1-ol</span>, <span class="green-bold">(Z)-11-hexadecenal</span>, <span class="blue-bold">(Z)-11-hexadecenyl acetate</span>) in our samples, <i>Nicotiana benthamiana leaves</i>.</p><br/><br/>
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<ul class="method">
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<li><a class="black-bold">Sample preparation:</a>
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<ul>
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    <li>Leaves must be immediately frozen in liquid nitrogen after removal from the plant to avoid any changes in their volatile profile.</li>
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    <li>Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap vial, avoiding defreezing.</li>
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    <li>EDTA and a saturated solution of CaCl2 are then added to the sample in order to inhibit enzymatic activity and nonenzymatic oxidation, thus stabilizing the sample.</li>
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    <li>Finally, samples are sonicated to favour the release of volatiles from the plant material.</li>
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</ul>
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</li>
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<li><a class="black-bold">Trapping of volatiles</a>: the SPME fiber is introduced through the septum into the headspace of the vial in order to ‘trap’ the volatiles from the sample. During the adsorption process the sample is heated under continuous agitation to favour the diffusion of volatiles from the sample.</li>
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<li><a class="black-bold">Desorption</a>: release of the volatiles from the fiber by thermal desorption in the injection port of the Gas chromatograph.</li>
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</ul><br/><br/>
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<p>Now the sample is ready to be analysed by <span class="blue-bold">GC-MS</span>.</p><br/><br/>
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<p>For further information <a class="blue-bold">check the detailed protocol</a>.</p><br/><br/>
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<a class="button-content" id="goto-left" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology"><strong>&larr; Go back to Methodology</strong></a>
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<a class="button-content" id="goto-right" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_analysis"><strong>Go to Sample Analysis &rarr;</strong></a></br></br></br>
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<p align="center"><strong>References</strong></p><br/>
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<div style="position: relative; left: 3%; width: 96%;"><ol>
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<li>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 </li><br/>
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<li>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.</li>
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<li>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.</li>
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<li>Gullan PJ, Cranston S (2014) The insects: An Outline of Enthomology. Wiley-Blackwell.</li>
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</ol><br/><br/>
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Revision as of 21:20, 17 October 2014

Project > Modules > Methodology > Electroantennogram



Sample Preparation: Headspace SPME


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.



solid_phase_extraction

Figure 1. Schematic representation of the headspace solid-phase microextraction adsorption/desorption. In the adsorption step the fiber is exposed in the headspace of a vial containing biological material, where volatile organic compounds (shown in red) are adsorbed by the fiber. In the desorption step, the fiber is introduced in the Gas Chromatograph injection port where the volatile compounds are released by thermal desorption.


We have used HS-SPME coupled to Gas Chromatography-Mass Spectrometry detection to analyse the presence of pheromones ((Z)-11-hexadecen-1-ol, (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate) in our samples, Nicotiana benthamiana leaves.



  • Sample preparation:
    • Leaves must be immediately frozen in liquid nitrogen after removal from the plant to avoid any changes in their volatile profile.
    • Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap vial, avoiding defreezing.
    • EDTA and a saturated solution of CaCl2 are then added to the sample in order to inhibit enzymatic activity and nonenzymatic oxidation, thus stabilizing the sample.
    • Finally, samples are sonicated to favour the release of volatiles from the plant material.
  • Trapping of volatiles: the SPME fiber is introduced through the septum into the headspace of the vial in order to ‘trap’ the volatiles from the sample. During the adsorption process the sample is heated under continuous agitation to favour the diffusion of volatiles from the sample.
  • Desorption: release of the volatiles from the fiber by thermal desorption in the injection port of the Gas chromatograph.


Now the sample is ready to be analysed by GC-MS.



For further information check the detailed protocol.



← Go back to Methodology Go to Sample Analysis →


References


  1. 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

  2. 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.
  3. 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.
  4. Gullan PJ, Cranston S (2014) The insects: An Outline of Enthomology. Wiley-Blackwell.




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