Figure 4. Glycosylation and phosphorylation of P. metricus (wild type) hexamerins. Both hexamerins are glycosylated, but only Hexamerin 2 is phosphorylated. A: Both hexamerins shift in size toward lower molecular weight after deglycosylation with PNGase F. B: The same gel stained with phosphorylation specific Pro-Q diamond stain (Invitrogen). Hexamerin 2 and vitellogenin (between 150 and 250 kDa standard bands) appear to be phosphorylated in P. metricus hemo- lymph. The + sign marks the deglycosylation enzyme PNGaseF. doi:10.1371/journal.pone.0026641.g004 observations difficult or impossible in the field. Honey bees, although easily observed and the target of the first insect genome project, are domesticated animals and have been selected for docility and honey production. Our work combines the assets of the Polistes model system with tools of genomics deployed in a field setting, augmenting the studies of Toth et al. and Hunt et al. [17,31,38,39] and further advancing the Polistes model system. Our small final sample sizes (11 hexamerin 2 and 11 gfp) could reflect several variables that can be addressed in future RNAi experiments. In order to provide maximum assurance that we treated only gyne-destined larvae, we conducted our experiment in late July and August, corresponding to the mid to late phase of gyne production. Because nest mortality is a normal demographic occurrence, a higher sample size could be obtained by beginning an experiment earlier in the phase of gyne production. To increase the number of nest boxes at the start of a season could also increase the final sample size. The frequency of nest foundings in our nest boxes by P. metricus (47/300) was unexpectedly lower than that of previous studies conducted in Missouri, USA. The four putative Hexamerin 1 peptide hits (8% sequence coverage) toward the Hexamerin 2 sequence in the MALDI experiment could be explained by some level of contamination or by sequence identity in these regions. Because the gene that encodes Hexamerin 1 is not identified, precise sequence similarity estimation between two hexamerins is currently infeasible. Previous studies of other hymenopteran species with sequenced genomes have reported relatively high sequence similarity of multiple hexamerins: up to 42% in A. mellifera [40] and 79% in the wasp Nasonia vitripennis [41]. We found that the two putative hexamerins of P. metricus are glycosylated. In Hexamerin 2, the putative N-glycosylation sites are located at asparagines 201 and 344. Glycosylation has been shown to have important stabilizing and structural roles in a related storage protein, arylphorin, in Antheraea pernyi silk moth [42]. Based on SDS- PAGE size-estimation, Hexamerin 1 (,77.7 kDa) lies within the RNAi in the Eusocial Wasp Polistes Figure 5. Validation of hexamerin 2 knockdown after larval feedings. RNA isolated from whole larvae (n = 18 per treatment type) was used for qRT-PCR. Compared to controls, hexamerin 2 dsRNA feeding elicited a significant reduction in hexamerin 2 transcript. Bars represent means 6 s.e.; asterisk = P,0.05. doi:10.1371/journal.pone.0026641.g005 typical hexamerin size scale of 75–90 kDa, whereas Hexamerin 2 is smaller than an average hexamerin with its molecular weight of ,68.5 kDa [40,41]. In addition to differences in size, only Hexamerin 2 appears to be phosphorylated. Phosphorylation can be critical for events ranging from signaling to subcellular protein localization [43,44]. Hexamerin-receptor interaction is regulated by receptor phosphorylation in rice moth Corcyra cephalonica [45], but effect of phosphorylation of hexamerin itself are currently unclear. The differences that we found between Hexamerins 1 and 2 in both size and phosphorylation suggest different roles for the two proteins. In a previous study using gel band quantification, Hexamerin 1 was significantly differentially expressed between worker-destined and gyne-destined larvae, but that experiment indicated no specific caste-related role for Hexamerin 2 [31]. In the present study we used dsRNA for the hexamerin 2 gene to specifically down- regulate production of Hexamerin 2 protein. The absence of a significant caste-related response to RNAi down-regulation of Hexamerin 2 confirms the inference of the previous study. However, our detailed statistical analysis suggests possible trends toward extended larval development time and reduced ovary development in knockdowns, which are characteristics of gynes [31,39]. The detailed statistical analysis is warranted, even though the statistical results per se are negative, because both positive and negative statistical outcomes are meaningful for interpretation of the study. Whereas dsRNA injection studies achieve significant knock- down of the target gene at low dosage [46,47], feeding studies generally require a higher dosage to achieve efficient knockdown. Effective knockdown in Polistes was achieved using a dosage of dsRNA consistent with feeding studies in honey bees [21,48] and termites [22]. Control (gfp) and hexamerin 2 larvae were fed equally with dsRNA without adverse phenotypic effects, thereby suggest- ing that the system will be compatible with future RNAi experiments. A next step, therefore, would be to continue investigation of roles played by hexamerins in P. metricus worker/ queen differentiation using hexamerin 1 RNAi in a full replication of the study we report here, once transcripts for the P. metricus hexamerin 1 gene have been identified. Such an investigation could test the possibilities of compensatory or opportunistic upregulation of Hexamerin 1 by Hexamerin 2. Compensation would be physiological – to have Hexamerin 1 substitute for Hexamerin 2 in functions that are not yet known. ‘Opportunistic’ would be that Hexamerin 1 increases because more building blocks are available if less Hexamerin 2 is reduced. In addition, methods such as microarray [38] and RNAseq can be employed to identify PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e26641