synthesized using primers that cover 520 bases in the open reading frame of the AF097553 template as previously described for gene targeting in honey bees (Wang et al 2010). The dsRNA was purified using phenol:choloform extraction and verified for size and purity by running on 1% agarose gels. The final concentration of dsRNA was 4–5 mg/ml. The sequence used to generate hexamerin 2 dsRNA is underlined in Figure 2. Larval sex determination We determined larval sex by genotyping 9 microsatellite loci that were previously developed from expressed sequence tag sequences [52]. We used 2 mg of total RNA for cDNA synthesis using SuperScript VILO cDNA Synthesis Kit (Invitrogen) and then used the cDNA for microsatellite genotyping. PCR reactions and fragment visualization were performed as previously described [31]. Verification of hexamerin 2 knockdown Fifth-instar larvae received 2 feedings per day for 2 days of either hexamerin 2 dsRNA or gfp dsRNA. Two days later, in the field, we processed single specimens as each was removed from its nest cell. Larval gut contents, called meconium, are contained within a chitinous peritrophic sac. The meconium volume is particularly large in fifth-instar larvae. To remove the meconium makes subsequent processing of the specimen easier and also obviates any possible RNA degradation. Using a pointed scalpel, we pierced the body and midgut walls and removed the meconium by grasping the peritrophic sac with fine-tipped forceps and quickly extracting it. The larva was then flash frozen in liquid N2. Specimens were further processed in the lab by homogenization and storage in Trizol reagent. We randomly selected 18 female larvae per treatment group for knockdown verification via real-time quantitative reverse- transcriptase PCR (qRT-PCR) using a one-step qRT-PCR kit (Ambion). Total RNA was isolated and treated with turbo DNase I (Ambion). Briefly, total RNA (25 ng/ml) was used as the template for reverse transcription. All qPCR reactions were run in triplicate using an ABI Prism 7500 (Applied Biosystems), and we analyzed the data with the Delta-Delta CT method [53] with rps8 as reference gene [17,54]. The hexamerin 2 forward and reverse primers were 59- AAACAGCATTATGGGCTCGT-39 and 59-GACGTTGAAC- GACAGCATT-39 respectively. For rps8, the forward and reverse primers were 59-GAAGCGAAAGCCTCTCAGAA-39 and 59- AGGCGCAGTGCTCTGTATTT-39. A negative control (without reverse transcriptase enzyme) was run for every sample to monitor DNA contamination. We used melt curve analysis to verify the absence of primer-dimers. Field rearing and in vivo dsRNA feedings We reared and monitored colonies of P. metricus using established protocols [30,55,56] (Fig. 7A). We placed three hundred nest boxes in weedy fields and at the margins of lawns and hay fields, all of which were near woodlands at sites owned and managed by North Carolina State University or the North Carolina Department of Agriculture in Wake Co., NC, within 10 km of the North Carolina State University (NCSU) campus. Forty-seven P. metricus nests were founded, all but one by a single foundress each. We worked only with single-foundress colonies. During the course of the field work we monitored colony development daily by removing the nest box top (Fig. 7B) and recording the content of each nest cell (empty, egg, larval instar, or cocoon) on a ‘nest map’ drawn on hexagon-imprinted paper. We collected colony data in the early mornings, generally before 9 am EDT, which enabled counting the number of adults present before daily foraging had begun. RNAi in the Eusocial Wasp Polistes We conducted all dsRNA treatments in the field using late 5th instar larvae in single-foundress colonies in situ in the nest boxes. Hexamerins 1 and 2 are not expressed in 1st through 4th instar larvae [49]. Instead, they are expressed in 5th (final) instar larvae and reach peak abundances at the larva to pupa molt [31,49]. Our RNAi treatments were applied to late 5th instar larvae to coincide with peak Hexamerin expression, thus our results are not a consequence of having treated larvae in a time window that might not yield a response signal. Only a single dsRNA type was used per nest. For treatments, tubes of dsRNA were placed on ice and transported to the field, where we mixed the dsRNA with 30% sucrose (Fig. 7C) immediately before feeding it to larvae using a standard laboratory pipette (Fig. 7D). At each feeding we gave each larva 15 ml of the dsRNA/sucrose mix, which they readily accepted. Each larva was treated morning and evening on 2 consecutive days. Each feeding contained 20 mg of dsRNA, therefore a total of 80 mg of dsRNA was administered to each larva. A total of 72 larvae from 10 nests were fed hexamerin 2 dsRNA, and a total of 56 from 8 nests were fed gfp dsRNA (Table S2). We identified larvae by location on the nest map. When a fed larva completed growth and spun its cocoon, we marked that cocoon with dots of TestorsH enamel (Fig. 7B inset). We continued nest mapping daily until all fed larvae in a nest had spun cocoons. When all fed larvae in a nest had spun cocoons, we transferred that colony to a rearing cage as described in [57]. Adults taken with the nest were anesthetized with CO2 and examined for wing wear. From 1 to 5 females with wing wear, which characterizes foundresses and workers [39], were marked with a dot of enamel on the metanotum and caged with the nest. We released any males and females with unworn wings, which were putative gynes [39]. We removed and discarded all unmarked cocoons and the untreated developing wasps in them as well as any subsequently cocooning individuals. Eggs and pre-cocooning larvae were undisturbed. We held caged colonies in a sheltered outdoor location at the NCSU Honey Bee Research Facility, where each colony was provided with ad libitum water and sucrose (rock candy) and a fresh mid-3rd instar Manduca sexta caterpillar daily. When all treated wasps had emerged from a nest, the remaining colony was returned to its nest box in the field and thereafter not disturbed. The field studies did not involve any endangered or protected species. No specific permits were required for the described field studies. Treatment bioassay We inspected caged colonies daily at mid-morning and identified newly-emerged wasps by nest map location prior to transporting them to the laboratory and placing each in an isolation cage. Isolation cages had wooden tops and bottoms ,8.5 cm on each side. Three sides of the cage were wire mesh (,3 mm openings) ,23 cm in height. The fourth side was closed with corrugated cardboard into which a small doorway was cut for daily maintenance. A small, empty, pre-emergence nest (7 to 14 cells) constructed in the preceding year by a P. metricus foundress near St. Louis, MO, was affixed to the underside of the top of each cage before the newly-emerged wasp was introduced. Most wasps spent time on those nests. Caged wasps were placed in growth chamber C25 in the NCSU Phytotron. The chamber contained incandescent lights on a 16L/ 8D cycle; fluorescent lights came on 2 h following and went off 2 h before the incandescent lights. Light intensity was 21 micromoles/ sec/m2 in the incandescent-only morning and evening periods and 225 micromoles/sec/m2 during the both-lights midday. Chamber temperature was 20uC at night and 30uC during full light, with a PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e26641