gradual ramp-up in the morning and ramp-down in the evening. We provided each isolation cage with a small plastic Petri dish containing sucrose (rock candy), a water source (a 1.5 ml microfuge tube plugged with cotton), and, daily, a single small M. sexta caterpillar: late 2nd-instar or early 3rd-instar. Caterpillar head capsules were crushed immediately before provisioning, which ensured that they did not crawl away. Cage maintenance, conducted daily at midday, consisted of replenishing the water (deionized), removing the remains of any uneaten caterpillars, and providing a fresh caterpillar. Time outside the chamber was no more than 2–3 min. Each day we repositioned cages within the chamber. Treatment response variables In a previous laboratory experiment with newly-emerged isolated P. metricus females, putative workers showed ovarian development whereas putative gynes did not [11]. Wasps entering ovary development could be expected to show higher rates of feeding from protein sources than wasps not entering ovary development. Because ovary development occurs in putative workers but not putative gynes, and because ovary development could reflect post-emergence feeding, we quantified both feeding rate and ovary development in order to test whether hexamerin 2 treatment might induce ovaries more worker-like than those of controls. Beginning the day after being introduced to the chamber, we scored each wasp daily for 12 days as to whether the caterpillar had been eaten. On the 13th day we removed each wasp from the chamber, chilled it ,10 min in a refrigerator, and removed it from its cage. We dissected ovaries (together with the digestive tract) by grasping the terminal segment of the metasoma with fine-point forceps and pulling. Removed tissue was placed in 100% EtOH in a labeled microfuge tube and stored at 220uC. Approximately 2 weeks after the last specimen had been prepared, we transferred the tissue samples to 70% EtOH and returned them to 220uC. Approximately 2 months later we dissected the tissue specimens in 70% EtOH under a dissecting microscope to separate the ovaries from other tissues. We then placed the ovaries in 70% EtOH in number-coded 0.2 ml microfuge tubes and held them at laboratory ambient temperature. Approximately 1 month later we digitally photographed each ovary using an Evolution MP Color camera on an Olympus SZX12 microscope with a 1.06 objective lens and the adjustable magnification set at 256. All images were captured in a single session with no adjustments to the apparatus. Four ovary images were selected as representing 4 development classes: 0 = ovaries filamentous with no discernable oocytes; 1 = oocytes discernable but small in size and somewhat translu- cent; 2 = one or more oocytes opaque (with yolk deposition); 3 = one or more large ova present (Fig. S3). We removed the selected images from the image set, and the remaining images were independently scored 0 to 3 by five non-biologists who compared each image to printed copies of Figure S3. For every ovary, we used the mean of these 5 objective scores for analyses. Scorers were blind to the identities of the specimens. We clipped the right forewing of each wasp at its base using iris scissors and taped it to a labeled microscope slide with clear tape. We then captured digital images of the wings with the same camera/microscope equipment as for ovaries but with a 0.56 objective lens and with the adjustable magnification set at 106. All images were captured in a single session with no adjustments to the equipment. We quantified images using ImageJ software. A linear measurement (in pixels) was taken from the proximal closure of the C cell to the distal posterior corner of the 2Rs cell (nomenclature RNAi in the Eusocial Wasp Polistes as in [58]). Wing wear by some wasps when in isolation precluded total wing length measurement. Measurements were blind with regard to which dsRNA a wasp had received. We converted pixel values to mm by quantifying a scale photographed at the same magnification. Statistical analyses The nests that received gfp and hexamerin 2 dsRNA treatment to verify hexamerin 2 knockdown were equally represented at two study sites. For RNAi validation, we collected gfp control larvae from six nests and putative hexamerin 2 knockdown larvae from four nests. Only a single dsRNA was administered in a single nest. Gene expression data were log transformed. We then used a main effects ANOVA in a model with mRNA level as the dependent variable and with qPCR plate (technical blocking factor) and treatment as the dependent variables. The dataset conformed to assumptions of ANOVA, as determined by Bartlett’s tests and normality plots of residuals. For analyses of individual development and adult bioassays, the gfp controls represented five nests at three locations, and the hexamerin 2 dsRNA treatment group represented four nests and three locations. The majority of the data (20 out of 22 wasps) came from two overlapping sites. To test whether treatment effects could be confounded by environmental influences or heterogeneity between location and nests, we first tested the effect of these two study sites on phenotypic characteristics, using a general linear model with location as random factor, treatment as a fixed effect, and ovary development, total developmental time, forewing length, and number of caterpillars eaten as dependent variables. All analyses were performed with Statistica 6.0. Supporting Information Figure S1 Amino acid alignment of the putative P. metricus hexamerin protein with other known insect hexamerin sequences. Polistes sequence was aligned with sequences of from other hymenopteran insects species, Apis meliferra (ABR45904), Nasonia vitripennis (XP_001607029) and Camponotus festinatus (CAB62053), and dipteran insects, Aedes aegypti (XP_001663961), Culex quinquefasciatus (XP_001843494) and Anopheles gambiae (XP_321434) using ClustalW version 1.82. The hemocyanin_N, (residues 29–151), hemocyanin_M domains (residues 158–415), and the hemocyanin_C, (residues 435–519) are all under-lined with dashed, solid and double-dashed lines respectively. Conserved residues among all taxa are highlighted in black bars, conserved substitutions among all taxa are highlighted in gray bars and semi-conserved substitutions are highlighted by light gray bars. Gaps in the alignment are indicated by dashes. (TIF) Figure S2 Electrospray mass spectrometric scan of Hexamerin 2 peptides. Peptide corresponding to dsRNA sequence LTTYFDQFDTSLNNGLVVESQK with mass 840.4 Da, indicated with the arrow, is only found in Hex2. (TIF) Figure S3 Ovary categories 0 to 4 that were used as comparators for scoring ovary development. (TIF) Primer sequences used for cloning P. metricus hexamerin Table S1 2. (DOC) Table S2 Data records for all treated specimens. Columns, left to right, are the specimen identifier, treatment, date of first feeding PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e26641