Synergistic Effects of Direct and Indirect Defences on Herbivore Egg Survival in a Wild

ELECTRONIC SUPPLEMENTARY MATERIAL

Synergistic effects of direct and indirect defences on herbivore egg survival in a wild crucifer

N.E. Fatouros et al.

Trichogramma species identification

DNA Extraction, PCR amplification and Electrophoresis for molecular species verification For the DNA extraction wasps were ground with a sterile glass pipette in 50μl 5% Chelex-100 and 4μ proteinase K (20mg/ml) and incubated for 6 hours at 56°C, followed by 1 min centrifugation, 10 min at 90°C and another centrifugation of 1 minute. The PCR was performed in a total volume of 50 μl which includes 1 μl DNA sample, 5 μl 5x Green GoTaq Buffer, 1,5 μl 25mM MgCl2, 0,5 μl 10mM dNTP’s, 0,12 GoTaq Polymerase 5U/ μl, 15,88 of sterile and distilled water and 0,5 μl of forward and reverse primers (ITS2-forward:’5-TGTGAACTGCAGGACACATG-3’ located in the 5.8S region of the rDNA; ITS2-reverse: 5’GTCTTGCCTGCTCTGAG-3’ [38]. The PCR cycling program was 3 min at 94°C followed by 33 cycles of 40 sec at 94°C, 45 sec at 55°C, 45 sec at 77°C and 10 min of 72°C after the last cycle. The gel electrophoresis was performed with a gel composed of 1% Agarose, 100ml TAE buffer and 3,5 μl 10mg/mL Ethidium Bromide. The gel was run with 4 μl marker (100bp Orange Ruler) and 10 μl PCR products.

Cloning, Sequencing and Alignments. Following electrophoresis, PCR products were purified with a Qiaquick Gel Extraction Kit according to the protocol. The cells were transformed with a heat shock according the pGEM®-T and pGEM®-T Easy Vector Systems protocol. The cells were plated on an agar medium that contained Trypton, Xgal, ampicillin and IPTG. The plates were incubated overnight at 37°C. The next day, the white colonies were picked up with sterile toothpicks from the plates. The toothpicks were then put into tubes containing 200 ml LB medium and 200ml Amp and put into a shaker to grow overnight at a temperature of 37°C with 250 rpm. After the incubation, a GeneElute Plasmid Miniprep Kit was used according to the protocol to purify the plasmid. The purified DNA was then sent to Eurofins MWG Operon (Germany) for sequencing the plasmid. The Trichogramma sequences were aligned with Geneious and matched against sequences present in GenBank.

Plant response to eggs

Figure S1. Linear regression of Pieris egg density and HR induction. Relationship between the number of eggs of Pieris spp. collected per plant and sampling day and the percentage of eggs inducing HR-like necrosis in B. nigra plants in 2011 (both generations). Numbers behind the dots indicate number of multiple data points. N data pairs = 50. Linear regression, p = 0.81.