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Orientierung von eiablagebereiten Manduca sexta Weibchen zu Pflanzendüften

Verhalten und chemische Analytik

Written by A. Gluschak

Paper category

Bachelor Thesis

Subject

Biology

Year

2011

Abstract

Bachelor Thesis: Plant volatiles analysis Headspace sampling to collect volatiles from the whole plant. A total of five plants of each species (D. wrightii, N. attennuata, and B. oleracea) were used. The plants were transferred to six 25-liter glass steel bottles, and 1.2 liters/min of charcoal filtered air was permanently pressed into them. The scent-laden air passes through the outlet in the side wall at a rate of 1 liter/min. Carbotrap C, B and X (Sigma Aldrich) 25 mg custom adsorption filters each are connected via short Teflon tubing. Odor collection started 30 minutes after the start of the dark phase and lasted for 7 hours. On the same day, empty tanks (Blanks) from 10:00 to 17:00 and collection of plant scents from 22:30 to 5:30. Between each collection of volatiles, thoroughly clean the cylinder with acetone and blow with clean air (push: 3 liters/minute, pull: 1.1 liters/minute, within 10 minutes). The adsorbent is eluted approximately 2 hours after the headspace collection is over. First, an internal standard containing 25 ml of dichloromethane (DCM) and 20 1-bromodecane (stock solution: 1 ng/) must be prepared. Each trap was flushed with internal standard in 400 DCM. The solution with each trap odor molecule is contained in a separate vial. Then concentrate the eluate to 50 μl under a gentle stream of nitrogen. Transfer the remaining part to the insert (volume: 100 ). After that, the eluate was concentrated again to 25 μl under a gentle stream of nitrogen. Finally, store the test at -80°C until analysis. Gas chromatography-mass spectrometry analyzes plant-scented compounds on a 7890A gas chromatograph (GC) (Agilent Technologies, CA, USA) (department: Evolutionary Neuroethology) operating in splitless mode. Keep the injection port at 230°C and inject 1 μl. A non-polar chromatography column (HP-5 MS ui & Innowax; 30 m, 0.25 mm ID, 0.25 μm film thickness; J&W Scientific, Folsom CA, USA) was used to operate at a constant He flow rate (1.1 ml/min). At the beginning, there was condensation for ten minutes in the cold trap at -60°C. Then it heats up to 12° per second. Therefore, the temperature is 210° after ten minutes. When injecting 1μl sample, the temperature is close to 230°. Then the GC column oven heats the system to 240° and keeps it for 5 minutes. The transfer line to the MS is maintained at 280°C, the MS is operating in electron impact mode (70 eV), the ion source is 230°C, the quadrupole is 150°C, and the mass scan range is 33 to 350 amu. Total ion chromatogram Recorded by an Agilent 5975C mass spectrometer connected to the GC. The nuclear compound is emitted by electrons, which are accelerated in an electric field. The difference is that electrons are deflected according to their mass in the magnetic field. The detector measures the deflection intensity. Data analysis First, the Kovats retention time index (KI) must be determined by saturation of a 10 ng alkane mixture. In this way, a safe analysis can be carried out. For each plant mass spectrometry (MS) report, subtract the blank MS report of the same trap and the blank MS report of the trap used on the same day. With the help of MSD Chem Station and NIST05 library (American National Institute of Standards and Technology; Gaithersburg, USA), the remaining compounds accepted as plant odors were analyzed, and the KI-value was calculated through MPI. Statistical online program (http://www.online-datenanalysis.de/Vorzeichenrangtest/2vS-Test.html) uses Wilcoxon two sample tests to check the difference between two odor sources in contact with dummy or abdominal bending in one run . In addition, the unpaired T test is applied through an online program (http://www.graphpad.com/quickcalcs/ttest1.cfm) to compare the five times (stationary phase, activation time, event time, and proximity time) to each other Various experiments. In the wind tunnel experiment, when D. wrightii headspace and clean air were provided, 52.5% of the test females first came into contact with fake leaves with the scent of D. wrightii during flight (Figure 2A; SD = 17,24). In addition, 14% of these moths bend their abdomen to show the intention to lay eggs (Table 1). In contrast, 10% of moths contacted the clean air dummy for the first time, and 6% of moths subsequently bent their abdomens. Thirty-one percent of the tested females were exposed to the dummy and N. attenuata odor source (Figure 2B; SD = 17,62), and 19% first contacted artificial plant leaves with clean air. In this experiment, only one insect responded to the bending of the abdomen (Table 1). Only a few insects (26%) first came into contact with a dummy with a B. oleracea odor source (Figure 2C; SD = 11,93), while 15% of insects first came into contact with a clean air dummy. When the odors of D. wrightii and N. attenuata appeared at the same time, more moths responded by contacting the leaves of alternative plants that were mainly D. wrightii odor sources (D. wrightii: 49%, N. attenuata: 17%, see Figure) .3A; SD = 12,01). 8% chose D. wrightii scented moths with curved abdomens (Table 1). When the odors from N. attenuata and B. oleracea were present at the same time, females showed no preference (Figure 3B; SD = 20,81). Twenty-two percent of the people who first came into contact with the dummy were related to the source of N. attenuata odor, and 24% of the people first came into contact with the dummy and B. oleraceaodour. In this experiment, only one insect responded to the bending of the abdomen (Table 1). In addition, all repeated contacts between each female and the two replacement leaves were counted, and the preference index was calculated on this basis (Figure 4, see Annex II for the original data). Read Less