title: Venom Y Kockdown assayed in Neobellieria bullata Rep2
design: Mated N. vitripennis females were hosted with S. bullata pupae and kept at 25C. Six days after hosting, the larvae of N. vitripennis were removed from the host and kept on 1XPBS plates during and after dsRNA injection [24]. dsRNA of VenYB was synthesized from genomic DNA using the MEGAScript RNAi Kit [Life Technologies, Grand Island NY], according to the manufacturer protocol. dsRNA targeting VenYB was injected at the posterior end of the larvae [24]. Control wasps were injected with dsRNA complimentary to the E. coli LacZ gene, since the N. vitripennis genome lacks a Lac operon. The larvae developed on 1XPBS plates at 25C until the second or third pupae stage when the N. vitripennis pupae were transferred into glass vials. After the enclosure, females from both groups were presented with Sarcophaga bullata hosts for eight hours to allow feeding and practice stinging, after which they were removed and starved overnight. The following morning, females were set individually on S. bullata pupae placed in drilled foam plugs to ensure exposure of only the anterior end of the host puparium for stinging and oviposition. After the wasps were removed, all S. bullata hosts had their anterior pupal cap removed to verify a sting site and remove any eggs laid by N. vitripennis. Hosts were stored at 25C and allowed to develop with a gel capsule (Medisa Inc.) covering their exposed anterior end to prevent dehydration. Hosts were sampled 72 hours after envenomation and stored in TRIzol Reagent (Ambion) at -80C. Extraction was performed as per manufacturers protocol (Ambion), and RNA quality and concentration was determined using an Agilent 2100 bioanalyzer. Three pools of five whole venom control hosts and VenYB knockdown hosts were used for TruSeq mRNA (Illumina) library construction and sequencing on an Illumina HiSeq2500 Sequencer at URGRC. Each library was sequenced on 1/12th of one lane.