Figure 1.
Colony cycle of an annual Polistes paper wasp, exemplified by P. metricus.
Foundress Phase. The annual cycle begins (arrow) when a female emerges from quiescence with undeveloped ovaries. Feeding at flowers enables ovary development, which precedes nest founding. Oviposition occurs immediately upon initiation of each nest cell by the now-maternal foundress (crown icon) that performs all maternal behaviors in addition to oviposition, including foraging, feeding larvae, and nest construction. Worker Phase. The first female offspring to emerge in early summer are workers (hard hat icon) that forage, feed larvae, and construct the expanding nest. The foundress transitions into a true social queen that limits her activities to oviposition and feeding larvae using foods brought to the nest by workers. Reproductive Phase. Female offspring emerging in mid-summer are non-working future foundresses called gynes (tiara icon). Males, not shown, emerge synchronously with gynes. Decline Phase. In late summer the queen and workers die. Gynes and males depart the nest, which now is empty and unattended. Quiescence Phase. Gynes and males mate and feed at fall flowers. With the decline in flower availability and onset of cooler weather, males die, and gynes enter a torpor-like quiescence in sheltered concealment. Workers live only from their emergence until the end of the reproductive phase. A gyne from the reproductive phase of one year has the potential to become a foundress and then queen the following year, thus the tiara to crown transition can represent single individuals that pass from gyne to foundress to queen. Larval treatments and sampling reported in this experiment were conducted in the Reproductive Phase of the colony cycle.
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 5′ 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.
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.
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 hemolymph. The + sign marks the deglycosylation enzyme PNGaseF.
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 ± s.e.; asterisk = P<0.05.
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 −). 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 − phenotypes differed significantly for time to cocoon.
Figure 7.
Field methods and dsRNA delivery.
A: “Wasp boxes” have lift-off lids and wire mesh across the open bottom. Boxes were put in place in springtime prior to natural nest establishment on the undersides of lids by P. metricus foundresses. The early foundress phase nest in this box is indicated by the arrow. B: A reproductive phase study nest on the underside of a box lid during the experiments. Newly-spun cocoons of larvae that had received dsRNA were marked with dots of paint (inset). C: dsRNA and sucrose solutions were carried to the field on ice and mixed immediately prior to larval treatments. D: dsRNA/sucrose solution was fed directly to the mouthparts of large 5th-instar larvae via micropipette. Larvae consumed the sample quickly, generally in ∼1 sec.