Illumina HiSeq 2500 sequencing; RNA-seq of three Spodoptera frugiperda tissues (Midgut, Fat body, Hemocytes), at 2 time-points (8h and 15h) after infestation by entomopathogenic nematodes Steinernema carpocapsae/Xenorhabdus nematophila
RNA-seq of three Spodoptera frugiperda tissues (Midgut, Fat body, Hemocytes), at 2 time-points (8h and 15h) after infestation by entomopathogenic nematodes Steinernema carpocapsae/Xenorhabdus nematophila
Spodoptera frugiperda larvae were bled and hemolymph was collected in anti-coagulant buffer (van Sambeek and Wiesner 1999). Hemocytes were recovered by a short centrifugation at 800 g for 1 min at 4°C. The hemocyte pellet was immediately flash-frozen. Then, larvae were dissected and fat bodies and midguts were extracted, rinsed with PBS, flash-frozen with liquid nitrogen in eppendorf tubes and conserved at -80°C until use.   Nematode production and storage Symbiotic Steinernema carpocapsae (strain SK27 isolated from Plougastel, France) were renewed on White traps (White 1927) after infestation of the wax moth Galleria mellonella (Lepidoptera : pyralidae) last larval stages. They were maintained in aerated Ringer sterile solution with 0.1 % formaldehyde at 8°C for two to four weeks to ensure optimal pathogenicity.  Insect rearing  Corn variant Spodoptera frugiperda (Lepidoptera : Noctuidae) larvae were reared on a corn-based artificial diet (Poitout 1970). They were maintained at 23°C +/- 1°C with a photoperiod of 16 h/8 h (light / dark) and a relative humidity of 40 % +/- 5 %. Galleria mellonella larvae were reared on honey and pollen at 28°C in dark.  Infestation experiments were processed at 23°C in 12-well culture plates. In each well, one second day sixth instar larva of S. frugiperda was placed on a filter paper (Whatman) with artificial corn-based medium. For infested larvae, 150 µL of Ringer sterile solution containing 150 S. carpocapsae IJs were introduced in each well. 150 µL of Ringer sterile solution was used for control larvae. To control the nematodes' efficacy, the survival of 12 larvae in both conditions was monitored for 48 hours in each experiment. After thawing, 1 mL of Trizol (Life technologies) was added and pooled organs were grounded with a TissueLyzer 85210 Rotator (Qiagen) with one stainless steel bead (3 mm diameter) at 30 Hz for 3 min. Grounded tissues were transferred in new eppendorf tubes and left at room temperature for 5 min then two hundred µL of chloroform (Interchim) were added. The preparations were homogenized and left at room temperature for 2 min. After a centrifugation at 15,000 g and 4°C for 15 min, the aqueous phase was transferred in new eppendorf tubes. Four hundred µL of 70% ethanol were added and nucleic acid extraction was immediately done with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Contaminating DNA was removed by the use of a Turbo DNA-freeTM kit (Life Technologies) according to the manufacturer's protocol. RNA yield and preparation purity were analyzed with a Nanodrop 2000 spectrophotometer (Thermo Scientific) by the measure of the ratios A260/A280 and A260/A230, respectively. RNA integrity was verified by agarose gel electrophoresis. RNA preparations were then conserved at - 80 °C. Library preparation and RNA sequencing were conducted by MGX GenomiX (IGF, Montpellier, France). Libraries were prepared with the TruSeq Stranded mRNA Sample preparation kit (Illumina). In brief, after a purification step with oligo(dT) magnetic beads, polyadenylated RNAs were chemically fragmented. A first cDNA strand was synthesized with random primers and SuperScript IV Reverse Transcriptase (Life Technologies) and the second strand was then synthesized. After the addition of single adenine nucleotides, indexed adapters were ligated to the cDNA ends. Adapters-ligated cDNAs were then amplified by PCR and libraries were validated on Fragment Analyzer with a Standard Sensitivity NGS kit (Advanced Analytical Technologies, Inc) and quantified by qPCR with a Light Cycler 480 thermal cycler (Roche Molecular diagnostics).
Experimental Factor: infect
PBS
Experimental Factor: time
15