To synchronize the insects, late third instar S. exigua larvae (L3) were selected the day before the feeding experiments. The following day, approx. 16 h after the selection, the newly moulted L4 larvae were separated and exposed, individually, to a dose of Vip3Aa that produced a 99% growth inhibition (111 ng/cm2; value obtained from toxicity experiments performed in our laboratory, data not shown). As a control, the filtered supernatant obtained from the E. coli control culture and diluted to the same degree as was required to dilute the Vip3Aa-containing supernatant, was used to feed the larvae. Larvae were exposed to the supplemented food for 8 h and 24 h. After these times, only larvae that had fed (as determined by observing the food bites) were selected for midgut dissection. Midguts of the larvae from each treatment were pooled for further processing.
Growth protocol
S. exigua larvae from the FRA colony kindly provided by M. López-Ferber (INRA, St. Christol les Alés, France) [33], were used in the experiments. The colony was reared at 25°C, with a relative humidity of 70%, and a photoperiod of 16 h:8 h (light: dark), on an artificial diet .
Extracted molecule
total RNA
Extraction protocol
The RNA from S. exigua midguts was purified using RNAzol reagent from Molecular Research Center, Inc. (Cincinnati, OH, USA), and purified using the RNAeasy Kit (Qiagen GmbH, Hilden, Germany) following the protocols provided by the manufacturers. The quality of RNA was assessed with an Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Nano protocol.
Label
Cy3
Label protocol
Agilent One-Color Spike-in Mix was added to the purified RNA and 600 ng of total RNA was used for complimentary RNA (cRNA) synthesis. Fluorescent labeling, fragmentation and hybridization were performed following the One-Color Microarray-Based Gene Expression Analysis (Quick-Amp labeling) protocol from Agilent.
Hybridization protocol
RNA labeling and hybridization, as well as array scanning and data extraction, were performed according to Agilent protocol, by the Microarray Analysis Service of the Principe Felipe Research Centre (CIPF), Valencia, Spain.
Scan protocol
S. exigua microarray chips were scanned using a G2505B Agilent scanner.
Description
Gene expression in the midgut of L4 larvae of Spodoptera exigua 8 hrs after treatment with VIP3A toxin T1_R9_8
Data processing
Data were extracted using Agilent Feature Extraction 9.5.1 software. Arrays were normalized using spike-ins and quantile normalization methods in Babelomics 4.3 software. Normalized signal intensities are given.
