RNAi in the Eusocial Wasp Polistes Figure 6. Median and 25–75% interquartile ranges for RNAi phenotypes. P. metricus larvae were assigned to a control group that received dsRNA for green fluorescent protein (gfp) or to a treatment group that received dsRNA against the hexamerin 2 gene to trigger RNA interference (hexamerin2 2). The wasps were monitored during post-treatment development and isolated for bioassays on the day they emerged as adults. A: The number of days from the start of treatment with dsRNA until cocoon spinning (time to cocoon). The significance indicator denotes Mann-Whitney U- test, P = 0.017, N = 11. This test statistic is significant only if multiple testing is not accounted for. B: The number of days between cocooning and adult emergence (time cocooned). C, D: Ovary development in 13 day-old adults as (C) a binary score (active or not – categories 1 and 2 of Fig. S3) and (D) scored as the 4 developmental classes of Figure S3. E: Length in mm of adult forewings from the proximal convergence of the C cell to the distal posterior corner of the 2Rs cell. F: Number of caterpillars eaten during the post-emergence isolation period. Control gfp and treatment hexamerin2 2 phenotypes differed significantly for time to cocoon. doi:10.1371/journal.pone.0026641.g006 candidate genes for other ecologically and evolutionarily impor- tant traits such as behavioral dominance, seasonal and nutritional affects on the colony cycle, photoperiodicity, and diapause. The genetic foundations of these traits can then be pursued using RNAi in experiments such as we report here. Ever-lower costs of whole genome sequencing using high throughput sequencing techniques, combined with improvements in annotation and comparative analysis methods, now allow genomic tools to be developed for many species. Efforts are currently underway to sequence genomes of two Polistes species: P. dominulus and P. canadensis (A. L. Toth, personal communication). Thus, soon there will be a substantial increase in genome sequence information for Polistes. The RNAi tool that we present here, therefore, is an extremely timely step forward that can enhance the use of paper wasps as model systems for the greater synthesis among gene expression, behavioral ecology, and evolutionary biology of social insects. In addition, although there are unique features of Polistes biology that make it particularly suitable for RNAi experiments in the field, our work suggests the possibility that RNAi is a tool that can be adapted for experiments on other species in natural environments. Methods Hexamerin sequencing Four partial EST sequences for hexamerin (contigs 45616, 45913, 42334 and 40833: http://stan.cropsci.uiuc.edu/454/ blast/waspblast.html) were available from the Polistes wasp EST data set to design primers. Total RNA isolated from 5th instar larvae was treated with DNaseI (Ambion), and 59 and 39 RACE were carried out using the GeneRacer Kit (Ambion). For cloning, we used different combinations of primers for 59 and 39 RACE (Table S1) in conjunction with the RACE primers provided in the PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e26641