RNAi in the Eusocial Wasp Polistes Figure 2. Nucleotide sequence of hexamerin 2 cDNA clone from P. metricus, along with the deduced protein sequence. The arrowhead indicates the signal cleavage site as predicted by SignalP (www.cbs.dtu.dk/services/SignalP). The underlined portion of the amino acid sequence corresponds to the dsRNA used for down-regulating hexamerin 2. The region corresponding to the 59 UTR (Untranslated Region) is shown with lowercase letters. The hexamerin 2 sequence shown is partial and does not include the C-terminal end (,50 amino acids). The GenBank accession number for the hexamerin 2 sequence is HQ661804. doi:10.1371/journal.pone.0026641.g002 hexamerin 2 knockdowns and gfp controls (Mann-Whitney U-test, P.0.05, N = 11, Fig. 6B, D–F). In contrast, days to cocoon and ovary development score (binary) were significantly different and suggestively different, respectively (Mann-Whitney U-test, P = 0.017; P = 0.094, N = 11, Fig. 6A, C). These results suggest that hexamerin 2 down-regulation could extend larval development and tentatively reduce ovarian development in adults. However, when corrected for multiple comparisons (Dunn-Sidak Bonferroni, alpha,0.009), neither effect could be considered as statistically reliable. Finally, we studied the phenotypic trait associations indepen- dent of treatment group identity. Adding to the significant negative correlation between days to cocoon and days cocooned (above), the data showed a significant negative relationship between total developmental time and ovary development (Pearson correlation, R = 20.526, P = 0.012, N = 22). This finding indicated that, in the dataset overall, individuals with a shorter total developmental time were more likely to have developed ovaries when scored as adults. Discussion Here we have reported the successful development of an RNA interference tool and characterization of its target protein for the primitively eusocial paper wasp Polistes metricus. Our most significant contribution to RNAi studies is that we then successfully deployed the RNAi tool in a field setting – that is, in a natural setting outside the laboratory. We did so in the context of an ecological test of caste differentiation in the eusocial wasp Polistes, which is a model system for evolutionary studies of insect sociality and behavioral ecology. The foundational aspects of our work included sequencing the hexamerin 2 gene, matching the cloned gene to the Hexamerin 2 protein product, and identification of the correspondence between the cloned gene and Hexamerin 2 by mass-spectrometry. Our principal biological finding – that Hexamerin 2 plays no role in worker/gyne differentiation – coincides with findings from previous studies using other investigative methodologies. Our research underscores that Polistes is a robust model system, having a rare convergence of assets. Paper wasps, with their 2 dimensional, bare nests allow access for observation and manipulation in their natural environment. As a result, they are a model that can be used to address questions regarding the structure and evolution of insect sociality. Most other social insects have been studied in artificial habitats. Ants and termites nest underground or within solid structures, thus making in-nest Figure 3. Mass-spectrometric spectra of Hexamerin 1 and Hexamerin 2, and the peptide hits of Hexamerin 2 against hexamerin sequence. The signal sequence in the N-terminus and the dsRNA in the C-terminus are underlined. A: MALDI-TOF/TOF spectra for Hexamerin 1. B: MALDI-TOF/TOF spectra for Hexamerin 2. C: MALDI-TOF/TOF peptide hits (grey boxes) and LC-MSMS hits (red text) for Hexamerin 2. A specifically inspected LC-MSMS peptide found in Hexamerin 2 data, is boxed with a red dashed line. doi:10.1371/journal.pone.0026641.g003 PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e26641