dsRNA was synthesized for hexamerin 2 and a control gene (gfp – green fluorescent protein), and these were used in a larval feeding experiment with naturally-founded P. metricus colonies in the field. We show that larval Hexamerin 2 can be successfully suppressed by RNAi and that the adult caste phenotype of Hexamerin 2 knockdowns is not significantly altered. These results as well as suggestive trends in our data support the proposition of diverging roles of the Hexamerin 1 and Hexamerin 2 products in caste development. Results Sequence of hexamerin 2 transcript and protein The P. metricus EST database contains four partial sequences, Contig45616, Contig45913, Contig42334, and Contig40833, that share homology with A. mellifera hexamerins. We used RACE to complete the cDNA ends, resulting in a single cDNA (Fig. 2) that overlaps all four contigs. The N-terminal sequence of the encoded protein constitutes a putative secretion signal peptide for an extracellular protein (www.cbs.dtu.dk/services/SignalP/), with cleavage predicted between residues 18 and 19 (amino acids VSA-EY, Fig. 2). The predicted mass of the mature protein is ,68.5 kDa, which is slightly lower than observed on SDS-PAGE gel. This apparent difference in size could be due to post- translational modifications attached to the protein sequence. Accordingly, we tested for N- and O-glycosylation and found potential N-glycosylation sites at asparagine residues at positions 201 and 344 (www.cbs.dtu.dk/services/NetNGlyc/). The cloned gene belongs to the hemocyanin family and has three conserved domains: hemocyanin_N (an all-alpha domain; residues 29–151), hemocyanin_M (which binds to copper and is necessary for the oxygen transport in the hemolymph; residues 158–415), and hemocyanin_C (an ig-like domain; residues 435– 519) [34]. The gene shows highest identity to A. mellifera hexamerin 70A (64% identity), while the degree of shared identity to other hexamerins is 55% to Nasonia vitripennis, 47% to Camponotus festinatus, and 44% to three mosquito sequences: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae (Fig. S1). Mass-spectrometry MALDI-TOF/TOF and LC-MSMS experiments revealed that Hexamerin 2 corresponds to the cloned gene based on 30%–40% sequence coverage, respectively (Fig. 3C). Hexamerin 1 had four hits against the sequence (YRGEEYLYGHK, LSNGLEK- VEDFDWSK, RIIYYYYR and EASYIIKAR; 8% sequence coverage), but got no hits in the subsequent LC-MSMS analysis. Both protein samples also contained unidentified non-trypsin, non- keratin peptides. MASCOT search score [35] for Hexamerin 2 LC-MSMS result against the cloned sequence was 207, with a sequence-based emPAI [36] score of 1.20. The peptide LTTYFDQFDTSLNNGLVVESQK that spanned the region of the dsRNA was, in particular, identified for Hexamerin 2 (Fig. S2). Deglycosylation and phosphorylation The two hexamerins have glycosylation in common, but they differ in the case of phosphorylation. Both Hexamerin 1 and Hexamerin 2 show reduced molecular weight on SDS-PAGE after treatment with PNGase F that removes N-linked glycogroups (Fig. 4A). The estimated size-shift was approximately 1.6 kDa for Hexamerin 1 and 1.8 kDa for Hexamerin 2, likely corresponding to the size of the glycogroup or total glycogroups attached to these proteins. Staining with phosphorylation-specific stain showed that of the two putative hexamerins only Hexamerin 2 was phosphor- ylated, and it was the only major phosphorylated protein in RNAi in the Eusocial Wasp Polistes addition to a putative vitellogenin in P. metricus hemolymph (Fig. 4B). This difference at the post-translational level might reflect the divergent effects of the proteins on P. metricus. Verification of hexamerin 2 down-regulation Compared to controls, larvae fed hexamerin 2 dsRNA (hexamerin 2 RNAi) showed a significant reduction in hexamerin 2 transcript levels (main affects ANOVA: F 1,32 = 6.258, p = 0.018) (Fig. 5). RNAi phenotype and trait associations Most of the 128 treated larvae (Table S2) could not be included in the final analysis. Forty-seven treated larvae were males, and 31 were cannibalized. Cannibalism occurs naturally and increases in frequency as colonies advance toward the end of their cycle [37]. Two colonies were destroyed (one by a mowing machine; one by Solenopsis invicta fire ants) for a loss of 10 specimens (6 gfp; 4 hexamerin 2). Eight larvae failed to feed or to spin cocoons. Because this is the first experiment of its kind we do not know if this was in response to treatments per se, disturbance during treatments, or some other cause. Seven larvae spun cocoons before receiving 4 treatment feedings (3 gfp; 4 hexamerin 2), and three adult wasps escaped. The remaining 22 larvae received 4 treatment feedings, spun cocoons, emerged as adult females, and were placed in the bioassay (11 total gfp from 5 nests; 11 total hexamerin 2 from 4 nests). We used these 22 specimens (Table S3) for all treatment analyses. Study site did not affect the wasps’ phenotype (F1,16 = 1.000, P = 0.500, N = 5, 15). Treatment was also not a significant factor in this corrected model that blocked for the effect of location on phenotypic traits (F1,16 = 0.040, P = 0.874, N = 10). No interaction was detected between study site and treatment (F1,16 = 0.042, P = 0.840). Next, we took into account that the total nine nests were split between the treatment categories. Using a nested design ANOVA to control for effects of nest within treatment, we found that neither treatment (F1, 13 = 0.008, P = 0.929, N = 11) nor nest (F7,13 = 0.528, P = 0.798, N = 4, 5) was significant. Thus, although the phenotypes showed some variability between nests (one-way ANOVA: F4, 20 = 34.116, P = 0.023, N = 6, excluding three nests with only 1 individual observation), the treatment groups were equally affected by inter-individual heterogeneity. We concluded that putative effects of treatment were not confounded by environmental influences or heterogeneity. To conserve degrees of freedom, therefore, factors location and nest were not used for blocking in subsequent analyses. We initially used a full, parametric model to analyze the phenotypic dataset. Total developmental time, ovary develop- ment, forewing length, and adult feeding were included as dependent variables in a one-way analysis of variance (ANOVA). Total developmental time was summed over the measurements of days to cocoon and days cocooned to avoid a colinearity problem caused by the two variables being significantly negatively associated in the dataset (Pearson correlation, R = 20.538, P = 0.001, N = 22). Egg number was not included due to insufficient data and variability (1 egg was laid by a gfp control wasp). The ANOVA suggested that hexamerin 2 RNAi did not alter the P. metricus phenotype. This result was independent of whether ovary development was coded as a binary variable (i.e., ovary being active or not: F4,17 = 2.259, P = 0.105, N = 11) or as 4 developmental classes (F4,17 = 1.961, P = 0.146, N = 11). The analysis conformed to assumptions of ANOVA, as determined by Bartlett’s test and Levene’s test on variances and normality plots of residuals. Next, we looked at each variable using a non-parametric approach. We found that days cocooned, ovary development (4 classes), forewing length, and adult feeding were similar between PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e26641