RNAi in the Eusocial Wasp Polistes Figure 7. Field methods and dsRNA delivery. A: ‘‘Wasp boxes’’ have lift-off lids and wire mesh across the open bottom. Boxes were put in place in springtime prior to natural nest establishment on the undersides of lids by P. metricus foundresses. The early foundress phase nest in this box is indicated by the arrow. B: A reproductive phase study nest on the underside of a box lid during the experiments. Newly-spun cocoons of larvae that had received dsRNA were marked with dots of paint (inset). C: dsRNA and sucrose solutions were carried to the field on ice and mixed immediately prior to larval treatments. D: dsRNA/sucrose solution was fed directly to the mouthparts of large 5th-instar larvae via micropipette. Larvae consumed the sample quickly, generally in ,1 sec. doi:10.1371/journal.pone.0026641.g007 kit. Following PCR, the amplified products were separated on 1% agarose gel, and DNA was extracted using the QIAquick Gel Extraction Kit (Qiagen). Thereafter, the PCR products were cloned into pCRH 4-TOPOH vector using the TOPO TA cloning kit (Invitrogen). Several clones were randomly picked and verified by sequencing. MALDI-TOF/TOF We identified the cloned hexamerin using peptide mass fingerprinting. To do so we ran dilution series of 0.2–0.8 ml P. metricus hemolymph from an adult gyne, a rich source of hexameric storage proteins [49], on a 4–20% gradient SDS-PAGE gel (Bio- Rad) followed by staining proteins with Coomassie blue (Bio-Rad). We then excised protein bands corresponding to Hexamerin 1 and Hexamerin 2 as identified by molecular weight [49] plus a clear gel region between the two proteins as a blank (control) sample. The samples were digested with trypsin and prepared for Ultra- flex MALDI-TOF/TOF (Bruker Daltonics) analysis according to existing protocols [50,51] using nC18 StageTip microcolumns [36]. We then processed spectra with FlexAnalysis (Bruker Daltonics) and targeted the annotated peaks for peptide mass fingerprint analyses with Mascot (Matrix Science; in house Mascot server) using all the NCBI databases and the P. metricus hexamerin sequence information we obtained by RACE. LC-MSMS We excised gel bands of Hexamerin1, Hexamerin 2 (4 gel pieces), and a blank sample as above. Peptides were prepared as above and separated by Ultimate 3000 nano-HPLC (Dionex Corporation, USA) using a nC18 reverse phase column coupled to a 4000 Q-Trap mass spectrometer (Applied Biosystems/MDS SCIEX, Concord, ON, Canada) and eluted as described elsewhere [51]. We used Analyst (AB SCIEX) for data processing and Mascot for data analysis. Mascot search was done against the P. metricus hexamerin sequence embedded in all the common NCBI databases. We set the significance threshold at 0.05, ion score minimum at 20, and included only hits with expected values ,0.1. A putative peptide corresponding to the size of the sequence LTTYFDQFDTSLNNGLVVESQK, located at the dsRNA region, was inspected in detail in the elution pattern of the Hexamerin 2 peptides. Deglycosylation and staining of phosphorylated proteins We tested Hexamerin 1 and Hexamerin 2 for post-translational modifications, glycosylation and phosphorylation, to gain protein- level insight and to make a comparison between the hexamerins. In order to test the presence of N-linked glycogroups, we treated 0.2 ml P. metricus hemolymph for 1 h at 37uC with 1 ml PNGase F and kit buffers (New England BioLabs), followed by a 10 min denaturation at 95uC. We treated a control sample similarly but with no PNGase F. We then ran samples on a 4–20% gradient gel (Bio-Rad) and stained them with Pro-Q Diamond (Invitrogen), which exclusively stains phosphorylated proteins. Finally, we visualized the gel in trans-UV light with Gel Doc Imager (Bio-Rad) prior to staining it with Coomassie blue (Bio-Rad) for detection of all proteins. dsRNA synthesis dsRNA was synthesized as described by Amdam et al. [24] using the AmpliScribe T7 transcription kit (Epicentre Biotechnologies) for bidirectional in vitro transcription of a plasmid DNA construct using gene specific primers with flanking T7 transcription initiation sites. For Hexamerin 2 dsRNA synthesis, we used the following forward and reverse primers fused with the T7 promoter sequence (underlined): 59-TAATACGACTCACTATAGGGCGACGGCTCTACAAGA- GGATAATC-39 and 59-TAATACGACTCACTATAGGGCGA- GAAATATTTGAAGTTCTCTTC-39. The resulting dsRNA prod- uct was 360 bp long. As an RNAi treatment control, gfp dsRNA, was PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e26641